Methods and compositions for diagnosis and treatment of meningitis

ABSTRACT

The present invention provides a method of identifying meningitis as either bacterial meningitis or aseptic meningitis in a subject, comprising: a) measuring the amount of complement C3, complement factor B, complement membrane attack complex (MAC) protein, complement C5b, complement C6, complement C7, complement C8, and/or complement C9 in a cerebrospinal fluid (CSF) sample obtained from the subject; and b) comparing the amount of complement C3, complement factor B, complement MAC protein, complement C5b, complement C6, complement C7, complement C8, and/or complement C9 measured in (a) with the amount of complement C3, complement factor B, complement MAC protein, complement C5b, complement C6, complement C7, complement C8, and/or complement C9 measured in a control sample, wherein an amount of complement C3, complement factor B, complement MAC protein, complement C5b, complement C6, complement C7, complement C8, and/or complement C9 measured in (a) that is greater than the amount of complement C3, complement factor B, complement MAC protein, complement C5b, complement C6, complement C7, complement C8, and/or complement C9 measured in the control sample identifies the meningitis in the subject as bacterial meningitis.

STATEMENT OF PRIORITY

This application is a divisional application of, and claims priority to,U.S. application Ser. No. 15/308,573, which is a 35 U.S.C. §371 nationalphase application of International Application Serial No.PCT/US2015/028881, filed May 1, 2015, which claims the benefit, under 35U.S.C. §119(e), of U.S. Provisional Application Ser. No. 61/988,025,filed May 2, 2014 and U.S. Provisional Application Ser. No. 62/116,213,filed Feb. 13, 2015, the entire contents of each of which areincorporated by reference herein.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant No. NS29719awarded by the National Institutes of Health. The U.S. Government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to methods and compositions fordifferentiating bacterial meningitis from aseptic meningitis in asubject by detecting changes in the levels of specific complementproteins in the cerebrospinal fluid (CSF) of the subject.

BACKGROUND OF THE INVENTION

Bacterial meningitis remains a major cause of morbidity and mortality,with a high incidence of residual neurological impairment. Earlydiagnosis and immediate onset of adequate antimicrobial treatment areessential for the survival of patients with bacterial meningitis.However, establishing the diagnosis of bacterial meningitis represents adifficult task in most cases, since clinical signs of acute meningitisare non-specific, and laboratory examinations of cerebrospinal fluid(CSF) often do not accurately differentiate between bacterial andaseptic meningitis. Accurate differentiation between bacterial andaseptic (e.g., viral) meningitis is difficult as both are inflammatorydiseases that elicit similar host defense responses and clinicalsymptoms. Differential diagnosis can be made on positive identificationof the bacteria from the cerebrospinal fluid of the affected individual.Unfortunately, it may take several days to grow and identify thebacteria and 25% of the time culture results are negative or equivocaleven though the patients have bacterial meningitis. Similar or greatererror rates affect nearly every laboratory parameter used for diagnosticpurposes.

Due to the beneficial effects of early therapy in bacterial meningitis,antibiotics are often started before etiologic diagnosis is established.As a consequence, a high number of patients with aseptic meningitisreceive unnecessary antibiotic treatment, leading to unnecessary and/orprolonged hospitalization, an increased financial burden to the healthcare system, and exposure of the patient to nosocomial infections anddisorders, as well as other hazards associated with hospitalization.

Few laboratory parameters in the cerebrospinal fluid determine bacterialmeningitis with absolute certainty, such as positive cerebrospinal fluidculture and Gram staining Although highly specific, these parametersshow very low sensitivities and are therefore not useful in ruling outbacterial infection. In addition to microbiological analysis,non-specific parameters in the cerebrospinal fluid are commonly used forthe differential diagnosis of bacterial versus aseptic meningitis, suchas total and differential cerebrospinal fluid leukocyte count,cerebrospinal fluid protein and glucose concentrations, CSF/serumglucose ratio, cerebrospinal fluid lactate and C-reactive proteinlevels. However, the diagnostic value of these parameters remainscontroversial, since their range of distribution overlaps widely inaseptic and bacterial cerebrospinal fluid. The current tests fordiagnosing bacterial meningitis take hours to days for completion andhave significant false-positive rates. Polymerase chain reaction (PCR)can be employed, but only if specialized equipment and trained labpersonnel are available, and the causative agent is in significantquantities in the spinal fluid. Hospital admission of aseptic meningitispatients is unnecessary, adds a significant financial burden to thehealth care system (estimated at $1-2 B/yr.), and increases the risk ofdangerous hospital-acquired infections. There is a critical need for aninexpensive rapid assay that can distinguish between bacterialmeningitis and aseptic meningitis.

The present invention overcomes previous shortcomings in the art byproviding methods and compositions for differentiating bacterialmeningitis from aseptic meningitis in a subject by detecting changes incomplement protein levels in CSF of the subject.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of identifyingmeningitis as either bacterial meningitis or aseptic meningitis in asubject in need thereof, comprising: a) measuring the amount ofcomplement C3, complement factor B, complement MAC (comprising,consisting essentially of or consisting of a complex of complement C5b,complement C6, complement C7, complement C8 and complement C9),complement C5b, complement C6, complement C7, complement C8 and/orcomplement C9 in a cerebrospinal fluid (CSF) sample obtained from thesubject; and b) comparing the amount of complement C3, complement factorB, complement MAC, complement C5b, complement C6, complement C7,complement C8 and/or complement C9 measured in (a) with the amount ofcomplement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8 and/or complement C9measured in a control sample, wherein an amount of complement C3,complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8 and/or complement C9 measured in (a) thatis greater than the amount of complement C3, complement factor B,complement MAC, complement C5b, complement C6, complement C7, complementC8 and/or complement C9 measured in the control sample identifies themeningitis in the subject as bacterial meningitis.

In another aspect, the present invention provides a method of diagnosingbacterial meningitis in a subject in need thereof, comprising: a)measuring the amount of complement C3, complement factor B, complementMAC, complement C5b, complement C6, complement C7, complement C8 and/orcomplement C9 in a cerebrospinal fluid (CSF) sample obtained from thesubject; and b) comparing the amount of complement C3, complement factorB, complement MAC, complement C5b, complement C6, complement C7,complement C8 and/or complement C9 measured in (a) with the amount ofcomplement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8 and/or complement C9measured in a control sample, wherein an amount of complement C3,complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8 and/or complement C9 measured in (a) thatis greater than the amount of complement C3, complement factor B,complement MAC, complement C5b, complement C6, complement C7, complementC8 and/or complement C9 measured in the control sample diagnosesbacterial meningitis in the subject.

A further aspect of the present invention is a method of carrying out atreatment regimen for a subject with meningitis or suspected of havingmeningitis, comprising: a) measuring the amount of complement C3,complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8 and/or complement C9 in a cerebrospinalfluid (CSF) sample obtained from the subject; b) comparing the amount ofcomplement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8 and/or complement C9measured in (a) with the amount of complement C3, complement factor B,complement MAC, complement C5b, complement C6, complement C7, complementC8 and/or complement C9 measured in a control sample, wherein an amountof complement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8 and/or complement C9measured in (a) that is greater than the amount of complement C3,complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8 and/or complement C9 measured in thecontrol sample identifies the meningitis as bacterial meningitis,indicating a treatment regimen of hospitalization and antibiotic therapyand wherein an amount of complement C3, complement factor B, complementMAC, complement C5b, complement C6, complement C7, complement C8 and/orcomplement C9 that is less than or equal to the amount of complement C3,complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8 and/or complement C9 in the control sampleidentifies aseptic meningitis, indicating a treatment regimen of nohospitalization and no antibiotic therapy; and c) carrying out theindicated treatment regimen.

In an additional aspect, the present invention provides a dry-stripcapable of wicking a fluid applied thereto by capillarity within thestrip, said strip comprising, in an upstream (at a first end) todownstream (at a second end) direction and in the following order: 1) asample-application zone, 2) a reaction zone, and 3) a detection zone,wherein said reaction zone comprises a non-immobilized labeled firstantibody specific against an epitope of a complement MAC protein, whichcan be complement C5b, complement C6, complement C7, complement C8and/or complement C9, effective to form therewith, a mobile complementMAC, which can be complement C5b, complement C6, complement C7,complement C8 and/or complement C9 protein/antibody complex, and saiddetection zone comprises an immobilized second antibody specific againstan epitope of a complement MAC protein, which can be complement C5b,complement C6, complement C7, complement C8 and/or complement C9 proteinin said complex, wherein said first and second antibodies are specificfor different epitopes on the same complement MAC protein, which can becomplement C5b, complement C6, complement C7, complement C8 and/orcomplement C9 protein and wherein, after application of a body-fluidsample to the sample-application zone at the first end, (i) samplemigrates in a downstream direction on the strip toward the reactionzone, (ii) complement MAC protein, which can be complement C5b,complement C6, complement C7, complement C8 and/or complement C9 proteinin the sample reacts with the first antibody previously present in thereaction zone to form a mobile, labeled complement MAC, which can becomplement C5b, complement C6, complement C7, complement C8 and/orcomplement C9 protein/antibody complex, (iii) the mobile, labeledcomplement MAC, which can be complement C5b, complement C6, complementC7, complement C8 and/or complement C9 protein/antibody complex migratestoward the detection zone at the second end, (iv) the mobile, labeledcomplement MAC, which can be complement C5b, complement C6, complementC7, complement C8 and/or complement C9 protein/antibody complex bindsthe immobilized second antibody previously present in the detectionzone, thereby immobilizing said complex in the detection zone.

Also provided herein is a lateral flow immunoassay device for detectinga marker, comprising: a membrane strip; a detecting antibody that bindsa first epitope of the marker; a test line comprising a capturingantibody that binds a second epitope of the marker; and a control linecomprising an antibody that binds a control analyte, wherein the markeris selected from the group consisting of complement protein C3,complement protein factor B, complement MAC, complement C5b, complementC6, complement C7, complement C8 and complement protein C9, as well asany combinations thereof.

In addition, the present invention provides a lateral flow immunoassaydevice comprising: a membrane strip; a first detecting antibody thathinds a first epitope of complement MAC protein, which can be complementC5b, complement C6, complement C7, complement C8 and/or complement C9; afirst test line comprising a first capturing antibody that binds asecond epitope of complement MAC protein, which can be complement C5b,complement C6, complement C7, complement C8 and/or complement C9; asecond detecting antibody that binds a first epitope of complement C3; asecond test line comprising a second capturing antibody that binds asecond epitope of complement C3; and at least one control linecomprising an antibody that binds a control analyte.

Further provided herein is a lateral flow immunoassay device comprising:a membrane strip; a first detecting antibody that binds a first epitopeof complement MAC protein, which can be complement C5b, complement C6,complement C7, complement C8 and/or complement C9; a first test linecomprising a first capturing antibody that binds a second epitope ofcomplement MAC protein, which can be complement C5b, complement C6,complement C7, complement C8 and/or complement C9; a second detectingantibody that binds a first epitope of complement factor B; a secondtest line comprising a second capturing antibody that binds a secondepitope of complement factor B; and at least one control line comprisingan antibody that binds a control analyte.

The present invention also provides a lateral flow immunoassay devicecomprising: a membrane strip; a first detecting antibody that binds afirst epitope of complement MAC protein, which can be complement C5b,complement C6, complement C7, complement C8 and/or complement C9; afirst test line comprising a first capturing antibody that binds asecond epitope of complement MAC protein, which can be complement C5b,complement C6, complement C7, complement C8 and/or complement C9; asecond detecting antibody that binds a first epitope of complement C3; asecond test line comprising a second capturing antibody that binds asecond epitope of complement C3; a third detecting antibody that binds afirst epitope of complement factor B, a third test line comprising athird capturing antibody that binds a second epitope of complementfactor B; and at least one control line comprising an antibody thatbinds a control analyte.

Furthermore, the present invention provides a method of monitoring asubject who is receiving treatment for bacterial meningitis, comprising:(a) obtaining serial samples of cerebrospinal fluid (CSF) from thesubject; (b) determining a level of complement MAC protein, which can becomplement C5b, complement C6, complement C7, complement C8 and/orcomplement C9; complement C3 and/or complement factor FB in each of saidsamples; (c) comparing the level of complement MAC protein, which can becomplement C5b, complement C6, complement C7, complement C8 and/orcomplement C9; complement C3 and/or complement factor FB in the serialsamples to detect a change in the level of complement MAC, which can becomplement C5b, complement C6, complement C7, complement C8 and/orcomplement C9; complement C3 and/or complement factor FB over time; and(d) modifying treatment of the subject, based on the results of thecomparing step (c).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Comparison of cost and timeframe between a standard diagnosticwork up for bacterial meningitis and an exemplary embodiment of thepresent invention, which is a lateral flow assay, shown in a cassetteconfiguration.

FIG. 2. Complement C3 and factor B (FB) concentrations in the CSF ofpatients with infectious meningitis or controls. C3 (Panel A) and FB(Panel B) CSF levels were measured by ELISA in 20 patients withconfirmed bacterial meningitis, 21 patients with aseptic meningitis and63 control patients. C3 and FB CSF levels in patients with bacterialmeningitis were significantly elevated compared to those with asepticmeningitis or to controls (p<0.001, Wilcoxon rank sum test for allcomparisons). The solid horizontal lines indicate the mean complementprotein level for each group. The dotted line is the mean±2 SD of theaseptic meningitis group and is used for differentiation betweenbacterial versus aseptic meningitis.

FIG. 3. C5b-9 (MAC) concentrations in the CSF of shunt/EVD patients withand without infection. MAC CSF levels were measured by ELISA in 3patients with confirmed bacterial infection and 24 patients withoutinfection. MAC CSF levels in patients with bacterial infection weresignificantly elevated compared to those without infection (p<0.0002,unpaired t-test). The line is the mean±2 SD of the infected group and isused for differentiation between the infected and uninfected groups.

FIG. 4. Changes in CSF MAC levels as a function of time in a patientwith a shunt infection. CSF MAC levels were quantitated using an ELISAspecific for soluble MAC (Quidel Corp., San Diego, Calif.). Samples werediluted as needed to obtain quantifiable data and duplicate samplesassayed according to the manufacturer's instructions.

FIG. 5. Changes in soluble CSF MAC levels in patients with confirmedbacterial, aseptic or cryptococcal infection. MAC CSF levels weremeasured by ELISA in 2 patients with confirmed bacterial meningitis (asassessed by bacterial culture), 73 aseptic patients (negative bybacterial culture) and 1 patient with confirmed cryptococcal infection.MAC CSF levels in patients with bacterial infection were significantlyelevated compared to aseptic patients with detectable MAC levels(p<0.0001, unpaired t-test). The dotted line represents the mean±2 SD ofdetectable MAC CSF levels in aseptic patients and is used fordiscrimination between bacterial and aseptic meningitis. Samples werediluted as needed to obtain quantifiable data and duplicate samples wereassayed according to the manufacturer's instructions.

FIG. 6. Changes in CSF MAC levels in patients with shunt infections,asymptomatic ventricular enlargement (VE), symptomatic ventricularenlargement (SVE, “shunt failure”) and normals. CSF MAC levels in normalpatients were undetectable by ELISA in 3 patients. Similarly, CSF MAClevels were essentially undetectable in asymptomatic VE patients (only 1in 8 patients had detectable MAC levels). In contrast, SVE patients hadelevated CSF MAC levels with MAC detectable in 22 of 35 patients, whileCSF MAC levels in 6 patients with confirmed bacterial infection weresignificantly elevated compared to SVE patients (p<0.0001, unpairedt-test). Samples were diluted as needed to obtain quantifiable data andduplicate samples were assayed according to the manufacturer'sinstructions.

FIG. 7. Changes in CSF C3 levels in patients with shunt infections,asymptomatic ventricular enlargement (VE), symptomatic ventricularenlargement (SVE, “shunt failure”) and normals. CSF C3 levels in normaland asymptomatic VE patients were low as assessed by ELISA. In contrast,SVE patients had elevated CSF C3 levels with C3 detectable in allpatients, while CSF C3 levels in 3 patients with confirmed bacterialinfection were significantly elevated compared to SVE patients (p<0.017,unpaired t-test). Samples were diluted as needed to obtain quantifiabledata and duplicate samples were assayed according to the manufacturer'sinstructions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is explained in greater detail below. Thisdescription is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein will be apparent to those skilled in the art in light of theinstant disclosure which do not depart from the instant invention.Hence, the following specification is intended to illustrate someparticular embodiments of the invention, and not to exhaustively specifyall permutations, combinations and variations thereof.

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination. Moreover, the present invention also contemplates thatin some embodiments of the invention, any feature or combination offeatures set forth herein can be excluded or omitted.

The present invention is based on the unexpected discovery thatbacterial meningitis can be differentiated from aseptic meningitis in asubject by detecting changes in the levels of specific complementproteins in the cerebrospinal fluid (CSF) of the subject. In aparticular embodiment, the present invention provides a point-of-care,lateral flow assay (LFA) based on bacterial, but not viral, induction ofspecific complement proteins. The LFA of this invention demonstrateshigh specificity and sensitivity for diagnosing bacterial meningitisusing complement biomarkers. The LFA of this invention offerssignificant advantages over conventional laboratory methods currently inuse, such as 1) ease of use, allowing essentially all medical personnelto perform the assay; 2) rapid, onsite (point-of-care) results availablein minutes in the emergency room (ER) or rural/remote clinics or carefacilities; 3) substantially reduced clinical laboratory costs; and 4)no specialized equipment, reagents or storage requirements. Thediagnostic tests of this invention permit a physician to initiateappropriate treatment regimens, resulting in substantial healthcare costsavings, e.g., by minimizing or preventing unnecessary medical treatmentand hospitalization (see, e.g., FIG. 1)

While the complement response is an important defense system againstdisease, several features make accurate measurement of complementproteins difficult, particularly in a clinically relevant time period.Known technologies require at least about an hour, and often two hoursor more to determine complement protein levels. During this time,significant deterioration of a patient's condition may be occurring thatdoes not visibly manifest until significant, or even irreversible,damage has occurred. Additionally, complement proteins are known to beeasily activated by handling and other experimental conditions that cansignificantly affect assay results. Also, the passage of time is asignificant factor in spontaneous complement activation, even withouthandling.

The present invention provides technologies that address thesepreviously unidentified sources of problems. For example, in someembodiments, the present invention provides methods in which relevantsteps are all performed within a restricted time period. According tothe present invention, such methods provide advantages includingminimizing spontaneous complement activation and, alternatively oradditionally, providing clinically relevant data within a time period,measured from initiation of sample collection from a subject, that issubstantially reduced as compared with standard methodologies. In someembodiments, assays of this invention are completed within a timeperiod, measured from initiation of sample collection from a subject,that is less than about 120 minutes or fewer, 75 minutes or fewer, 60minutes or fewer, about 50 minutes or fewer, about 40 minutes or fewer,about 30 minutes or fewer, about 20 minutes or fewer, about 10 minutesor fewer, or about 5 minutes or fewer or about 3 minutes or fewer. Thereis currently no test, assay or protocol available that would allow sucha rapid turnaround time, therefore informing physicians in real timeregarding diagnosis, treatment and/or further testing. Thus, the presentinvention provide methods that are substantial improvements over thecurrent protocols for identifying and/or diagnosing subjects of thisinvention and over the current standard of care and treatment of suchsubjects.

Complement is notoriously fastidious and can become activated by virtueof standard analysis procedures (handling, storage, and exposure toforeign materials that contact C3 during analysis). Complement is veryeffective at lysing invading microbes and initiating the wound healingresponse at sites of injury. This effectiveness is due in part to theability of C3 to be activated by foreign materials such as bacterialcell wall components. While this property is useful in directing animmune response to new foreign pathogens, this same property presentsformidable challenges to experimental and diagnostic study. Materialssuch as plastics used in sample handling, manipulation of the sampleitself, and improper storage conditions can also trigger complementactivation. The more processing and handling steps required to perform agiven assay, the more false positives can be expected, due to activationof complement by virtue of the assay itself. Such false positivescomplicate traditional testing and render current testing methodsunsuitable for use in directing patient care in near real-time.

The complement system comprises more than 40 serum and cellular proteinsand plays important roles in innate and adaptive immunity. There arefour major pathways of complement activation. The classical pathway isprimarily activated by immune complexes, specifically IgG/IgM antibodiesbound to antigen. Other activators include lipopolysaccharide, myelin,polyanionic compounds, C reactive protein (CRP), and microbial DNA andRNA. The lectin pathway is activated by polysaccharides withfree-mannose group and other sugars common to fungi and bacteria. Thealternative pathway is mediated by direct C3 activation by “foreign”substances that often include microbial cell wall components. All threemajor pathways of complement activation converge on the central proteincomplement component 3 (C3). C3 is a central mediator of inflammationand is activated by most factors that cause inflammation. The classicalpathway is typically triggered by immune complexes, which are complexesof antigen bound with antibodies, generally belonging to the IgM or IgGisotypes. Immune complexes in turn bind to complement component C1,which is comprised of C1q, C1r, and C1s. The binding of C1q to anantibody-antigen complex triggers activation of C1r and C1s. ActivatedC1s then cleaves component C4 to produce C4a and C4b. C4b is capable ofcovalent attachment to cell surfaces, although only about five percentdoes so. The remaining 95 percent reacts with water to form a soluble,activated C4b. Complement component 2 can then associate with C4b, whichafter which it is activated by C1s to C2a and C2b. C4b and C2a combineto form C4bC2a, the classical pathway (CP) C3 convertase.

The CP C3 convertase cleaves C3 to form C3a and C3b. Like activated C4b,C3b can covalently bind to cell surfaces or react with H₂O and stay insolution. Activated C3b has multiple roles. By itself, it can serve asan opsonin to make the decorated cell or particle more easily ingestedby phagocytes. In addition, C3b can associate with C4bC2a (the CP C3convertase) to form a C5 convertase. The complex, termed C4bC2aC3b istermed the CP C5 convertase. Alternatively, C3b can form the core ofanother C3 convertase called the alternative pathway (AP) C3 convertase.

The alternative pathway (AP) is another mechanism by which C3 can becomeactivated. It is typically activated by targets such as microbialsurfaces and various complex polysaccharides and other materials. Thisalternative pathway can also be initiated spontaneously by the cleavageof the thioester bond in C3 by a water molecule to form C3(H₂O). C3(H₂O)binds factor B, which allows factor D to cleave factor B to Ba and Bb.Bb remains associated with C3(H₂O) to form C3(H₂O)Bb complex, which actsas a C3 convertase and cleaves C3, resulting in C3a and C3b.

C3b formed either via this process or via the classical or lectinpathways binds to targets (e.g., on cell surfaces) and forms a complexwith factor B, which is subsequently cleaved by factor D and form Bb,resulting in C3bBb, which is termed the alternative pathway (AP) C3convertase. Binding of another molecule of C3b to the AP C3 convertaseproduces C3bBbC3b, which is the AP C5 convertase.

The lectin complement pathway is initiated by binding of mannose-bindinglectin (MBL) and MBL-associated serine protease (MASP) to carbohydrates.The MBL1 gene (known as LMAN1 in humans) encodes a type 1 integralmembrane protein localized in the intermediate region between theendoplasmic reticulum and the Golgi complex. The MBL2 gene encodes thesoluble mannose-binding protein found in serum. In the human lectinpathway, MASP1 and MASP2 are involved in proteolysis of C4 and C2,leading to C3 convertase, which lead to production of a C5 convertase asdescribed above for the CP.

C5 convertase generated via any of these three pathways cleaves C5 toproduce C5a and C5b. C5b then binds to C6, C7, and C8, which catalysespolymerization of C9 to form the C5b-9 membrane attack complex (MAC).The assembling MAC inserts itself into target cell membrane, forming apore delineated by a ring of C9 molecules. MAC formation causes celllysis of invading microbes, MAC formation on host cells can also causelysis, but not necessarily. Sublytic amounts of MAC on the membrane ofcells may affect cell function in a variety of ways. The small cleavageproducts C3a, C4a, and C5a are anaphylatoxins and mediate multiplereactions in the acute inflammatory response. C3a and C5a are alsopotent chemotactic factors that attract immune system cells such asneutrophils and macrophages into the area of crisis.

The extrinsic protease pathway is a more recently characterizedcomplement activation pathway that directly activates C3 and C5 throughthe enzymatic action of one or more activated coagulation enzymesincluding thrombin, plasmin and activated factors IX, X and XII.Activation of C3 and 5 through this pathway generates functionallyactive C3a, C3b, C5a and C5b in the absence of the canonical C3 and C5convertases formed through activation of the other three complementpathways.

Complement component C3 is useful as a general alert biomarker that thebody is responding to some form of physiological crisis, such as injury,infection, or other disease process. Similarly, complement component C9is a unique biomarker in the cerebrospinal fluid by virtue of itsability, in association with complement proteins C5b-C9 in the MACcomplex, to lyse susceptible bacterial strains that may cause bacterialmeningitis. Complement-mediated (C9) lysis results in the release ofbacterial cell wall and intracellular components, which directlyactivate innate immune inflammatory mechanisms that contribute to theseverity of meningitis. Complement has been associated with a widevariety of diseases, including lupus, arthritis, intracranialhemorrhage, diabetes, multiple sclerosis, heart disease, and age-relatedmacular degeneration. In many cases, the severity of disease correlateswith the level of complement activation. In some cases, complement canplay a role in disease pathology. In these cases, the body is not ableto successfully control the cause of inflammation, which spreads fromlocal site of injury to systemic inflammation. Complement activation candirectly damage tissue or do so indirectly by over-activating cells andrecruiting immune cells that in turn cause tissue destruction. Examplesof over activation include anaphylactic shock, multiple organ failure(MOF), acute respiratory distress syndrome (ARDS), and systemicinflammatory response syndrome (SIRS).

The assays and methods of the present invention provide severaladvantages over previous complement assays and methods known in the art.For example, the instant assays and methods are suitable forpoint-of-care (POC) use, producing results in a matter of minutes,rather than hours. The rapid return of results allows a clinician to actin near real-time to direct patient care. The assays and methods of thisinvention are easy to use and do not require the availability of alaboratory or a skilled lab technician. Additionally, the assays andmethods of this invention require fewer handling steps, and thusminimize complement activation due to handling and processing, whichleads to false positive test results.

Thus, in one aspect, the present invention provides a method ofidentifying meningitis in a subject (e.g., a subject in need thereof) aseither bacterial meningitis or aseptic meningitis, comprising: a)measuring the amount of complement C3, complement factor B, complementMAC, complement C5b, complement C6, complement C7, complement C8, and/orcomplement C9 in a cerebrospinal fluid (CSF) sample obtained from thesubject; and b) comparing the amount of complement C3, complement factorB, complement MAC, complement C5b, complement C6, complement C7,complement C8, and/or complement C9 measured in (a) with the amount ofcomplement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8, and/or complement C9measured in a control sample, wherein an amount of complement C3,complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8, and/or complement C9 measured in (a) thatis greater than the amount of complement C3, complement factor B,complement MAC, complement C5b, complement C6, complement C7, complementC8, and/or complement C9 measured in the control sample identifies themeningitis in the subject as bacterial meningitis.

In another aspect, the present invention provides a method of diagnosingbacterial meningitis in a subject (e.g., a subject, in need thereof),comprising: a) measuring the amount of complement C3, complement factorB, complement MAC, complement C5b, complement C6, complement C7,complement C8, and/or complement C9 in a cerebrospinal fluid (CSF)sample obtained from the subject; and b) comparing the amount ofcomplement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8, and/or complement C9measured in (a) with the amount of complement C3, complement factor B,complement MAC, complement C5b, complement C6, complement C7, complementC8, and/or complement C9 measured in a control sample, wherein an amountof complement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8, and/or complement C9measured in (a) that is greater than the amount of complement C3,complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8, and/or complement C9 measured in thecontrol sample diagnoses bacterial meningitis in the subject.

A further aspect of the present invention is a method of carrying out atreatment regimen for a subject with meningitis or suspected of havingmeningitis (e.g., a subject in need thereof), comprising: a) measuringthe amount of complement C3, complement factor B, complement MAC,complement C5b, complement C6, complement C7, complement C8, and/orcomplement C9 in a cerebrospinal fluid (CSF) sample obtained from thesubject; b) comparing the amount of complement C3, complement factor B,complement MAC, complement C5b, complement C6, complement C7, complementC8, and/or complement C9 measured in (a) with the amount of complementC3, complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8, and/or complement C9 measured in a controlsample, wherein an amount of complement C3, complement factor B,complement MAC, complement C5b, complement C6, complement C7, complementC8, and/or complement C9 measured in (a) that is greater than the amountof complement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8, and/or complement C9measured in the control sample identifies the meningitis as bacterialmeningitis, indicating a treatment regimen of hospitalization andantibiotic therapy and wherein an amount of complement C3, complementfactor B, complement MAC, complement C5b, complement C6, complement C7,complement C8, and/or complement C9 that is less than or equal to theamount of complement C3, complement factor B, complement MAC, complementC5b, complement C6, complement C7, complement C8, and/or complement C9in the control sample identifies aseptic meningitis, indicating atreatment regimen of no hospitalization and no antibiotic therapy; andc) carrying out the indicated treatment regimen.

Nonlimiting examples of other treatment regimens for a subjectidentified as having bacterial meningitis according to the methodsdescribed herein include modifying the duration, type and/or amount ofantibiotic therapy; administering ancillary therapies with intravenousfluids, etc.; modifying the duration and/or amount of dexamethasonetherapy; administering anti-complement therapy and/or anti-inflammationtherapy in combination with or instead of antibiotic therapy; and/ormodifying the duration and/or amount of ancillary therapy,anti-complement therapy and/or anti-inflammation therapy. Othertreatment regimens that could be modified include the need to perform CTor MRI imaging of the brain (for identification of infarction, abscess,hydrocephalus and other conditions) and repeated CSF analysis in thecase of resistant and/or complicated infections.

Many inhibitors of complement are known in the art and suitable for usewith the methods of the present invention. In some embodiments, theinhibitor of complement is selected from the group consisting of naturalcomplement inhibitors and derivatives thereof, compstatin and analogsthereof, anti-membrane attack complex (MAC) antibodies, anti-C3antibodies, anti-C5 antibodies, C3a receptor antagonists, and C5areceptor antagonists. Examples of additional complement inhibitors canbe found, for example, in Emlen et al. “Therapeutic complementinhibition: new developments” Semin. Thromb. Hemost. 36(6):660-68(2101); Wagner et al. “Therapeutic potential of complement modulation”Nat. Rev. Drug Discov. 9(1):43-56 (2010); and Ricklin et al.“Complement-targeted therapeutics” Nat. Biotechnol. 25(11):1265-75(2007), the contents of each of which are incorporated by referenceherein in their entireties.

Nonlimiting examples of other treatment regimens for a subjectidentified as having aseptic meningitis according to the methodsdescribed herein include reducing the duration of hospitalization and/oradministration of antibiotics, ancillary therapies, dexamethasonetherapy, anti-complement and anti-inflammatory therapies, the need forbrain imaging and frequency of contact with a treating physician.

The methods of this invention can also be used to modify standardprotocols for diagnosing bacterial meningitis. Currently, the standarddiagnostic workup includes a Gram stain, glucose measurement, proteinmeasurement, white blood cell (WBC) count, culture and possibly PCR, allon a CSF sample obtained from a subject. The methods of this inventioncan be carried out on a CSF sample from a subject as an initial test andif an elevated level of individual components complement C5b, complementC6, complement C7, complement C8, complement C9, the membrane attackcomplex, (MAC) consisting of C5b-C9, complement C3 and/or complementfactor B is detected in the CSF sample, thereby differentiatingmeningitis in the subject as bacterial meningitis, subsequent tests mayinclude, for example, a Gram stain and/or culture to identify thebacterial pathogen that is causing the bacterial meningitis, e.g., inorder to determine the best antibiotic regimen.

In further aspects, the present invention provides a method ofidentifying meningitis as either bacterial meningitis or asepticmeningitis in a subject in need thereof, comprising: a) measuring theamount of complement C5b, complement C6, complement C7, complement C8,complement C3, complement factor B, complement membrane attack complex(MAC) and/or complement C9 in a cerebrospinal fluid (CSF) sampleobtained from the subject; and b) comparing the amount of complement C3,complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8, and/or complement C9 measured in (a) withthe amount of complement C3, complement factor B, complement MAC,complement C5b, complement C6, complement C7, complement C8, and/orcomplement C9 measured in a control sample, wherein an amount ofcomplement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8, and/or complement C9measured in (a) that is greater than an amount of complement C3,complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8 and/or complement C9 measured in thecontrol sample identifies the meningitis in the subject as bacterialmeningitis.

Also provided herein is a method of identifying meningitis as eitherbacterial meningitis or aseptic meningitis in a subject in need thereof,comprising measuring the amount of complement C3, complement factor B,complement membrane attack complex (MAC), complement C5b, complement C6,complement C7, complement C8 and/or complement C9 in a cerebrospinalfluid (CSF) sample obtained from the subject, wherein an amount ofcomplement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8 and/or complement C9measured in (a) that is greater than a threshold amount of complementC3, complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8 and/or complement C9 identifies themeningitis in the subject as bacterial meningitis.

Further provided herein is a method of diagnosing bacterial meningitisin a subject in need thereof, comprising: a) measuring the amount ofcomplement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8 and/or complement C9 in acerebrospinal fluid (CSF) sample obtained from the subject; and b)comparing the amount of complement C3, complement factor B, complementMAC, complement C5b, complement C6, complement C7, complement C8 and/orcomplement C9 measured in (a) with the amount of complement C3,complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8 and/or complement C9 measured in a controlsample, wherein an amount of complement C3, complement factor B,complement MAC, complement C5b, complement C6, complement C7, complementC8 and/or complement C9 measured in (a) that is greater than the amountof complement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8 and/or complement C9measured in the control sample diagnoses bacterial meningitis in thesubject.

The present invention additionally provides a method of diagnosingbacterial meningitis in a subject in need thereof, comprising measuringthe amount of complement C3, complement factor B, complement MAC,complement C5b, complement C6, complement C7, complement C8 and/orcomplement C9 in a cerebrospinal fluid (CSF) sample obtained from thesubject, wherein an amount of complement C3, complement factor B,complement MAC, complement C5b, complement C6, complement C7, complementC8 and/or complement C9 measured in (a) that is greater than a thresholdamount of complement C3, complement factor B, complement MAC, complementC5b, complement C6, complement C7, complement C8 and/or complement C9diagnoses bacterial meningitis in the subject. Additional steps, whichcan be carried out in some embodiments, include treating the subjectwith antibiotics and/or other therapeutic agents, hospitalizing thesubject, etc.

In further embodiments, the present invention provides a method ofdiagnosing an infection associated with an indwelling shunt and/orextra-ventricular device in a subject in need thereof, comprising: a)measuring the amount of complement C3, complement factor B, complementMAC, complement C5b, complement C6, complement C7, complement C8 and/orcomplement C9 in a cerebrospinal fluid (CSF) sample obtained from thesubject; and b) comparing the amount of complement C3, complement factorB, complement MAC, complement C5b, complement C6, complement C7,complement C8 and/or complement C9 measured in (a) with the amount ofcomplement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8 and/or complement C9measured in a control sample, wherein an amount of complement C3,complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8 and/or complement C9 measured in (a) thatis greater than the amount of complement C3, complement factor B,complement MAC, complement C5b, complement C6, complement C7, complementC8 and/or complement C9 measured in the control sample diagnoses aninfection associated with an indwelling shunt and/or extra-ventriculardevice in the subject. This method allows for the identification of aninfection associated with an indwelling shunt and/or extra-ventriculardevice in a subject that may not be showing symptoms of such aninfection and/or for whom a source of infection has not been determinedor is difficult to determine. Employing the methods of this inventioncan inform a physician regarding whether treatment is needed and/or whattreatment approach to take. By using the methods described herein, aphysician can have information regarding whether the subject has aninfection within a much shorter period of time than would occuraccording to current protocols for diagnosis (e.g., waiting for culturedata). Thus, by using the methods of this invention, the subject canreceive treatment sooner if an infection is identified or the subjectcan be spared unnecessary treatment (e.g., with antibiotics), which maybe prescribed because an infection is suspected but not yet verified(e.g., by culture results).

A method is also provided herein of diagnosing an infection associatedwith an indwelling shunt and/or extra-ventricular device in a subject inneed thereof, comprising: measuring the amount of complement C3,complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8 and/or complement C9 in a cerebrospinalfluid (CSF) sample obtained from the subject, wherein an amount ofcomplement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8 and/or complement C9measured in (a) that is greater than a threshold amount of complementC3, complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8 and/or complement C9 diagnoses an infectionassociated with an indwelling shunt and/or extra-ventricular device inthe subject.

In further embodiments, the present invention provides a method ofdiagnosing an autoimmune disorder, traumatic brain injury and/or strokein a subject in need thereof, comprising: a) measuring the amount ofcomplement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8 and/or complement C9 in acerebrospinal fluid (CSF) sample obtained from the subject; and b)comparing the amount of complement C3, complement factor B, complementMAC, complement C5b, complement C6, complement C7, complement C8 and/orcomplement C9 measured in (a) with the amount of complement C3,complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8 and/or complement C9 measured in a controlsample, wherein an amount of complement C3, complement factor B,complement MAC, complement C5b, complement C6, complement C7, complementC8 and/or complement C9 measured in (a) that is greater than the amountof complement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8 and/or complement C9measured in the control sample diagnoses an autoimmune disorder,traumatic brain injury and/or stroke in the subject.

A method is also provided herein of diagnosing an autoimmune disorder,traumatic brain injury and/or stroke in a subject in need thereof,comprising: measuring the amount of complement C3, complement factor B,complement MAC, complement C5b, complement C6, complement C7, complementC8 and/or complement C9 in a cerebrospinal fluid (CSF) sample obtainedfrom the subject, wherein an amount of complement C3, complement factorB, complement MAC, complement C5b, complement C6, complement C7,complement C8 and/or complement C9 measured in (a) that is greater thana threshold amount of complement C3, complement factor B, complementMAC, complement C5b, complement C6, complement C7, complement C8 and/orcomplement C9 diagnoses an autoimmune disorder, traumatic brain injuryand/or stroke in the subject.

Furthermore, the present invention provides a method of identifying asubject as having an increased risk of developing an infectionassociated with an indwelling shunt and/or extra-ventricular device,comprising: a) measuring the amount of complement C3, complement factorB, complement MAC, complement C5b, complement C6, complement C7,complement C8 and/or complement C9 in a cerebrospinal fluid (CSF) sampleobtained from the subject; and b) comparing the amount of complement C3,complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8 and/or complement C9 measured in (a) withthe amount of complement C3, complement factor B, complement MAC,complement C5b, complement C6, complement C7, complement C8 and/orcomplement C9 measured in a control sample, wherein an amount ofcomplement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8 and/or complement C9measured in (a) that is greater than the amount of complement C3,complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8 and/or complement C9 measured in thecontrol sample identifies the subject as having an increased risk ofdeveloping an infection associated with an indwelling shunt and/orextra-ventricular device. By employing the methods of this invention, asubject can be identified who would benefit from medical interventioneven if the subject is not manifesting or showing symptoms of a problemor complication or inflammatory condition and/or infection associatedwith an indwelling shunt and/or extra-ventricular device. Such a subjectcould be treated prophylactically (e.g., with antibiotics, steroids,anti-inflammatory agents, etc.). A culture of the subject's CSF can becarried out to determine if the subject has an infection and if theculture results identify an infection, the subject can be treated forthe infection. A subject identified according to the methods describedherein as having an improperly functioning indwelling shunt and/orextra-ventricular device can undergo flushing of the shunt and/ordevice, removal or replacement of the shunt and/or device, etc. Asubject identified according to the methods as described herein canundergo imaging analyses and/or other testing to look for changes inventrical size, bleeding in the brain, etc. Thus, the benefit of themethods of this invention is that a subject can be identified as asubject in need of such testing and/or medical intervention when thereare no other indicators that the subject is in such need. There iscurrently no test or protocol available to identify such subjects and/orto carry out tests and/or treatments based on information about thelevels of complement proteins in the CSF of such subjects.

In addition, the present invention provides a method of identifying asubject as having an increased risk of developing an infectionassociated with an indwelling shunt and/or extra-ventricular device,comprising measuring the amount of complement C3, complement factor B,complement MAC, complement C5b, complement C6, complement C7, complementC8 and/or complement C9 in a cerebrospinal fluid (CSF) sample obtainedfrom the subject, wherein an amount of complement C3, complement factorB, complement MAC, complement C5b, complement C6, complement C7,complement C8 and/or complement C9 measured in (a) that is greater thana threshold amount of complement C3, complement factor B, complementMAC, complement C5b, complement C6, complement C7, complement C8 and/orcomplement C9 measured in the control sample identifies the subject ashaving an increased risk of developing an infection associated with anindwelling shunt and/or extra-ventricular device.

Also provided herein is a method of guiding a treatment for bacterialmeningitis in a subject in need thereof, comprising: a) measuring theamount of complement C3, complement factor B, complement MAC, complementC5b, complement C6, complement C7, complement C8 and/or complement C9 ina cerebrospinal fluid (CSF) sample obtained from the subject prior toadministration of the treatment for bacterial meningitis; b)administering the treatment to the subject; c) measuring the amount ofcomplement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8 and/or complement C9 in acerebrospinal fluid (CSF) sample obtained from the subject at one ormore time points after (b); d) guiding the treatment of the subject forbacterial meningitis using the measurement(s) of (c) such that anincrease or no change in the amount(s) measured in (c) relative to theamount(s) measured in (a) leads to a subsequent enhancement of thetreatment, and a decrease in the amount(s) measured in (c) relative tothe amount(s) measured in (a) leads to no change or a subsequentreduction of the treatment.

Further provided herein is a method of guiding a treatment for aninfection associated with an indwelling shunt and/or extra-ventriculardevice in a subject in need thereof, comprising: a) measuring the amountof complement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8 and/or complement C9 in acerebrospinal fluid (CSF) sample obtained from the subject prior toadministration of the treatment for the infection associated with anindwelling shunt and/or extra-ventricular device; b) administering thetreatment to the subject; c) measuring the amount of complement C3,complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8 and/or complement C9 in a cerebrospinalfluid (CSF) sample obtained from the subject at one or more time pointsafter (b); d) guiding the treatment of the subject for the infectionassociated with an indwelling shunt and/or extra-ventricular deviceusing the measurement(s) of (c) such that an increase or no change inthe amount(s) measured in (c) relative to the amount(s) measured in (a)leads to a subsequent enhancement of the treatment, and a decrease inthe amount(s) measured in (c) relative to the amount(s) measured in (a)leads to no change or a subsequent reduction of the treatment.

In an additional aspect, the present invention provides a method ofguiding a treatment for an autoimmune disorder, traumatic brain injuryand/or stroke in a subject in need thereof, comprising: a) measuring theamount of complement C3, complement factor B, complement MAC, complementC5b, complement C6, complement C7, complement C8 and/or complement C9 ina cerebrospinal fluid (CSF) sample obtained from the subject prior toadministration of the treatment; b) administering the treatment to thesubject; c) measuring the amount of complement C3, complement factor B,complement MAC, complement C5b, complement C6, complement C7, complementC8 and/or complement C9 in a cerebrospinal fluid (CSF) sample obtainedfrom the subject at one or more time points after (b); d) guiding thetreatment of the subject for an autoimmune disorder (e.g., multiplesclerosis (MS), systemic lupus erythematosis (SLE) rheumatoid arthritis(RA)), traumatic brain injury and/or stroke (including inflammatoryconditions and/or disorders associated with the brain and centralnervous system, brain abscess, central nervous system injury) using themeasurement(s) of (c) such that an increase or no change in theamount(s) measured in (c) relative to the amount(s) measured in (a)leads to a subsequent enhancement of the treatment, and a decrease inthe amount(s) measured in (c) relative to the amount(s) measured in (a)leads to no change or a subsequent reduction of the treatment.

Additionally provided herein is a method of treating a subject with aninfection associated with an indwelling shunt and/or extra-ventriculardevice, comprising: a) measuring the amount of complement C3, complementfactor B, complement MAC, complement C5b, complement C6, complement C7,complement C8 and/or complement C9 in a cerebrospinal fluid (CSF) sampleobtained from the subject; b) comparing the amount of complement C3,complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8 and/or complement C9 measured in (a) withthe amount of complement C3, complement factor B, complement MAC,complement C5b, complement C6, complement C7, complement C8 and/orcomplement C9 measured in a control sample, wherein an amount ofcomplement C3, complement factor B, complement MAC, s complement C5b,complement C6, complement C7, complement C8 and/or complement C9measured in (a) that is greater than the amount of complement C3,complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8 and/or complement C9 measured in thecontrol sample identifies an infection in the subject associated withthe indwelling shunt and/or extra-ventricular device; and c) treatingthe subject identified in (b) for the infection associated with theindwelling shunt and/or extra-ventricular device.

Furthermore, the present invention provides a method of treating asubject with an infection associated with an indwelling shunt and/orextra-ventricular device, comprising: a) measuring the amount ofcomplement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8 and/or complement C9 in acerebrospinal fluid (CSF) sample obtained from the subject, wherein anamount of complement C3, complement factor B, complement MAC, complementC5b, complement C6, complement C7, complement C8 and/or complement C9measured in (a) that is greater than a threshold amount of complementC3, complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8 and/or complement C9 identifies aninfection in the subject associated with the indwelling shunt and/orextra-ventricular device; and b) treating the subject identified in (a)for the infection associated with the indwelling shunt and/orextra-ventricular device.

In an additional embodiment, the present invention provides a method oftreating a subject with an improperly functioning indwelling shuntand/or extra-ventricular device, comprising: a) measuring the amount ofcomplement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8 and/or complement C9 in acerebrospinal fluid (CSF) sample obtained from the subject; b) comparingthe amount of complement C3, complement factor B, complement MAC,complement C5b, complement C6, complement C7, complement C8 and/orcomplement C9 measured in (a) with the amount of complement C3,complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8 and/or complement C9 measured in a controlsample, wherein an amount of complement C3, complement factor B,complement MAC, complement C5b, complement C6, complement C7, complementC8 and/or complement C9 measured in (a) that is greater than the amountof complement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8 and/or complement C9measured in the control sample identifies an improperly functioningindwelling shunt and/or extra-ventricular device; and c) treating thesubject identified in (b) to correct the improperly functioningindwelling shunt and/or extra-ventricular device.

Also provided herein is a method of treating a subject with animproperly functioning indwelling shunt and/or extra-ventricular device,comprising: a) measuring the amount of complement C3, complement factorB, complement MAC, complement C5b, complement C6, complement C7,complement C8 and/or complement C9 in a cerebrospinal fluid (CSF) sampleobtained from the subject, wherein an amount of complement C3,complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8 and/or complement C9 measured in (a) thatis greater than a threshold amount of complement C3, complement factorB, complement MAC, complement C5b, complement C6, complement C7,complement C8 and/or complement C9 identifies an improperly functioningindwelling shunt and/or extra-ventricular device; and b) treating thesubject identified in (a) to correct the improperly functioningindwelling shunt and/or extra-ventricular device.

A further embodiment of this invention provides a method of identifyinga treatment regimen for a subject with bacterial meningitis, comprising:a) measuring the amount of complement C3, complement factor B,complement MAC, complement C5b, complement C6, complement C7, complementC8 and/or complement C9 in a cerebrospinal fluid (CSF) sample obtainedfrom the subject; b) comparing the amount of complement C3, complementfactor B, complement MAC, complement C5b, complement C6, complement C7,complement C8 and/or complement C9 measured in (a) with the amount ofcomplement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8 and/or complement C9measured in a control sample, wherein an amount of complement C3,complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8 and/or complement C9 measured in (a) thatis greater than the amount of complement C3, complement factor B,complement MAC, complement C5b, complement C6, complement C7, complementC8 and/or complement C9 measured in the control sample identifies themeningitis as bacterial meningitis, identifying a treatment regimen ofhospitalization and antibiotic therapy and wherein an amount ofcomplement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8 and/or complement C9 that isless than or equal to the amount of complement C3, complement factor B,complement MAC, complement C5b, complement C6, complement C7, complementC8 and/or complement C9 in the control sample identifies asepticmeningitis, identifying a treatment regimen of no hospitalization and noantibiotic therapy; and c) carrying out the identified treatmentregimen.

The present invention also includes a method of monitoring a subject whois receiving treatment for bacterial meningitis, comprising: a)obtaining serial samples of cerebrospinal fluid (CSF) from the subjectprior to initiation of treatment and during treatment; b) measuring alevel of complement C5b, complement C6, complement C7, complement C8complement C9, complement C3, complement MAC and/or complement factor Bin each of said samples; c) comparing the level of complement C5b,complement C6, complement C7, complement C8 complement C9, complementC3, complement MAC and/or complement factor B in the serial samples todetect a change in the level of complement C5b, complement C6,complement C7, complement C8 complement C9, complement C3, complementMAC and/or complement factor B over time; and (d) modifying treatment ofthe subject, based on the results of the comparing.

The present invention further provides a method of monitoring a subjectwho is receiving treatment for an infection associated with anindwelling shunt and/or extra-ventricular device, comprising: a)obtaining serial samples of cerebrospinal fluid (CSF) from the subjectprior to initiation of treatment and during treatment; b) measuring alevel of complement C5b, complement C6, complement C7, complement C8complement C9, complement C3, complement MAC and/or complement factor Bin each of said samples; c) comparing the level of complement C5b,complement C6, complement C7, complement C8 complement C9, complementC3, complement MAC and/or complement factor B in the serial samples todetect a change in the level of complement C5b, complement C6,complement C7, complement C8 complement C9, complement C3, complementMAC and/or complement factor B over time; and (d) modifying treatment ofthe subject, based on the results of the comparing step.

Additionally provided herein is a method of monitoring the status of asubject with an indwelling shunt and/or extra-ventricular device,comprising: a) obtaining serial samples of cerebrospinal fluid (CSF)from the subject; b) measuring a level of complement C5b, complement C6,complement C7, complement C8 complement C9, complement C3, complementMAC and/or complement factor B in each of said samples; c) comparing thelevel of complement C5b, complement C6, complement C7, complement C8,complement C9, complement C3, complement MAC and/or complement factor Bin the serial samples to detect a change in the level of complement C5b,complement C6, complement C7, complement C8 complement C9, complementC3, complement MAC and/or complement factor B over time; and (d)implementing and/or initiating and/or continuing and/or modifying and/ordiscontinuing treatment of the subject, based on the results of thecomparing step.

In some embodiments of the methods of the present invention, only theamount of complement MAC in the CSF sample is measured.

In some embodiments of the methods of the present invention, only theamount of complement C5b in the CSF sample is measured.

In some embodiments of the methods of the present invention, only theamount of complement C6 in the CSF sample is measured.

In some embodiments of the methods of the present invention, only theamount of complement C7 in the CSF sample is measured.

In some embodiments of the methods of the present invention, only theamount of complement C8 in the CSF sample is measured.

In some embodiments of the methods of the present invention, only theamount of complement C9 in the CSF sample is measured.

In some embodiments of the methods of the present invention, only theamount of complement C3 in the CSF sample is measured.

In some embodiments of the methods of the present invention, only theamount of complement factor B in the CSF sample is measured.

In some embodiments of the methods of the present invention, the amountof complement MAC and the amount of complement C9 in the CSF sample aremeasured.

In some embodiments of the methods of the present invention, the amountof complement MAC and the amount of complement C3 in the CSF sample aremeasured.

In some embodiments of the methods of the present invention, the amountof complement MAC and the amount of complement factor B in the CSFsample are measured.

In some embodiments of the methods of the present invention, the amountof complement C9 and the amount of complement C3 in the CSF sample aremeasured.

In some embodiments of the methods of the present invention, the amountof complement C9 and the amount of complement factor B in the CSF sampleare measured.

In some embodiments of the methods of the present invention, the amountof complement MAC, the amount of complement factor B and the amount ofcomplement C3 in the CSF sample are measured.

In some embodiments of the methods of the present invention, the amountof complement C9, the amount of complement factor B and the amount ofcomplement C3 in the CSF sample are measured.

In some embodiments of the methods of the present invention, the step ofmeasuring can be carried out with a point of contact (POC) rapid assaysystem.

In some embodiments of the methods of the present invention, the step ofmeasuring can be carried out with a lateral flow system.

In some embodiments of the methods of the present invention, the step ofmeasuring can be carried out with an immunoassay.

In some embodiments of the methods of the present invention, the step ofmeasuring can be carried out with an enzyme-linked immunosorbent assay(ELISA).

The mean level of complement C3 in a CSF sample of a control (e.g.,healthy or non-infected subject) is 2.5 micrograms/ml. In subjects withbacterial meningitis, the mean level of complement C3 in a CSF sample is48 micrograms/ml. In the methods of the present invention, a referencelevel of complement C3 in a CSF sample is about 10 micrograms/ml.Therefore, a complement C3 level in a CSF sample of greater than 10micrograms/ml is increased relative to control and identifies a subjectwith meningitis as having bacterial meningitis rather than asepticmeningitis. A complement C3 level in a CSF sample of less than 10micrograms/ml identifies a subject with meningitis as having asepticmeningitis rather than bacterial meningitis.

The mean level of complement factor B (FB) in a CSF sample of a control(e.g., healthy or non-infected subject) is 0.5 micrograms/ml. Insubjects with bacterial meningitis, the mean level of complement FB in aCSF sample is 16 micrograms/ml. In the methods of the present invention,a reference level of complement C3 in a CSF sample is about 1.0microgram/ml. Therefore, a complement FB level in a CSF sample ofgreater than 1.0 microgram/ml is increased relative to control andidentifies a subject with meningitis as having bacterial meningitisrather than aseptic meningitis. A complement FB level in a CSF sample ofless than 1.0 microgram/ml identifies a subject with meningitis ashaving aseptic meningitis rather than bacterial meningitis.

The level of complement C9 in a CSF sample of a control (e.g., healthyor non-infected subject) can be in a range of about 200 ng/ml to about500 ng/ml. In the methods of the present invention, a reference level ofcomplement C9 in a CSF sample is about 500 ng/ml. Therefore, acomplement C9 level in a CSF sample of greater than 500 ng/ml isincreased relative to control and identifies a subject with meningitisas having bacterial meningitis rather than aseptic meningitis. Acomplement C9 level in a CSF sample of less than 500 nanograms/mlidentifies a subject with meningitis as having aseptic meningitis ratherthan bacterial meningitis.

In some embodiments, the level of complement MAC in a CSF sample or acontrol (e.g., healthy or non-infected subject or subject without anindwelling shunt and/or extra-ventricular device) is less than about 10ng/ml.

In some embodiments, the level of complement MAC in a CSF sample of asubject with shunt failure and/or a complication associated with anindwelling shunt and/or a subject at increased risk of developing aninfection associated with an indwelling shunt and/or extra-ventriculardevice can be, for example, in the range of about 20 ng/ml to about 100ng/ml. Thus, in some embodiments, a threshold value for identifying sucha subject can be a complement MAC level in CSF of greater than 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 75, 100, 125, 150,200 ng/ml but less than about 240, 245, or 250 ng/ml.

In some embodiments, the level of complement MAC in a CSF sample of asubject with a bacterial infection (e.g., bacterial meningitis, abacterial infection associated with an indwelling shunt orextra-ventricular device) can be, for example, greater than 250 ng/ml.Thus, in some embodiments, a threshold value for identifying such asubject can be a complement MAC level in CSF of greater than 200, 225,250, 300, 350, 400, 450, 500, 1000, 2000, 3000, or 4000 ng/ml.

In the methods of this invention, a threshold value can also bedetermined as a percent increase over a control value, e.g., 5%, 10%,15%, 20%, 25%, 30%, 35% 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%,200%, 250%, 300%, 400%, 500% or even 1000% greater than or increasedover a control value.

Nonlimiting examples of an antibody to complement C3 that can beemployed in the methods and assays of this invention include theantibodies listed in Table 1 herein, as well as any other antibody tocomplement C3 now known or later identified. These antibodies can beused in the methods and assays of this invention in any combination ofC3.

Nonlimiting examples of an antibody to complement factor B that can beemployed in the methods and assays of this invention include an antibodyfrom Hycult identified as complement factor B/Bb, Human, mAb M13/12,HM2256; an antibody from Comptech identified as Goat Anti-Human FactorB, Catalog No. A235; an antibody from Abcam identified as an Anti-FactorB antibody (ab182924; goat polyclonal to Factor B); an antibody fromAbcam identified as an Anti-Factor B antibody (ab72658; rabbitpolyclonal to Factor B); and an antibody from Abcam identified asAnti-Factor B antibody (ab190494; goat polyclonal to Factor B), as wellas any other antibody to complement factor B now known or lateridentified. These antibodies can be used in the methods and assays ofthis invention in any combination of FB antibodies. In some embodiments,the antibody can bind Bb.

Nonlimiting examples of an antibody to complement C9 that can beemployed in the methods and assays of this invention include apolyclonal rabbit anti-C9 produced in mouse against the peptide sequenceCMPIPVSREEQEQHYPIPID. This C9 antibody cross reacts with humancomplement C9. Other examples of an antibody to complement C9 that canbe employed in the methods and assays of this invention include anantibody from Hycult identified as C9, Human, mAb X197, HM2111; anantibody from Hycult identified as C9 neoantigen, Human, mAb WU 13-15,HM2264; an antibody from Comptech identified as Goat Anti-Human C9,Catalog No. A226; an antibody from Quidel identified as Goat anti-humanC9 #A310; an antibody from Quidel identified as Mouse anti-human C9#A223; an antibody from Abcam identified as Anti-C9 antibody (ab118902;rabbit polyclonal to C9); an antibody from Abcam identified as Anti-C9antibody (ab92690; rabbit polyclonal to C9); and an antibody from Abcamidentified as Anti-C9 antibody (ab53896; sheep polyclonal to C9), aswell as any other antibody to complement C9 now known or lateridentified. These antibodies can be used in the methods and assays ofthis invention in any combination of C9.

Nonlimiting examples of an antibody to complement MAC that can beemployed in the methods and assays and devices of this invention includean antibody to C5, C5b, C6, C7, C8, C9 and any combination thereof. Anantibody to complement MAC can be an antibody that binds C5, C5b, C6,C7, C8 and/or C9 when these respective complement proteins are presentin the complement MAC. For example, an antibody to complement MAC can bean antibody that binds to a neo-epitope on complement C9 that is presentwhen complement C9 is part of the complement MAC. An antibody tocomplement MAC can include an antibody that binds to a neo-epitopepresent on any of the complement proteins C5, C5b, C6, C7, C8 or C9 whenthese proteins are part of the complement MAC. Assays and devices ofthis invention can be employed to detect and/or quantitate any of theseantibodies in any combination.

The antibodies to C3, antibodies to FB antibodies to complement MAC andantibodies to C9 as described herein can be employed in the methods andassays of this invention in any combination of C3, FB, MAC and C9antibodies.

Furthermore, in embodiments of this invention employing antibodies, theantibodies that bind complement C9, the antibodies that bind C3, theantibodies that bind MAC and the antibodies that bind FB are notsubstantially cross-reactive. The antibodies employed in the methods andassays of this invention can be monoclonal antibodies or polyclonalantibodies, in any combination.

In some embodiments of the methods of this invention, only the amount ofcomplement C9 in the CSF sample is measured. In some embodiments, onlythe amount of complement C3 in the CSF sample is measured. In someembodiments, only the amount of complement factor B in the CSF sample ismeasured. In some embodiments, the amount of complement C9 and theamount of complement C3 in the CSF sample are measured. In someembodiments, the amount of complement C9 and the amount of complementfactor B in the CSF are measured. In some embodiments, the amount ofcomplement C9 and the amount of complement C3 and the amount ofcomplement factor B in the CSF are measured. In some embodiments,additional complement proteins in the CSF sample can be measuredaccording to the methods described herein, in any combination with, orindependent of, measurement of complement C9, complement C3 and/orcomplement factor B. Thus, nonlimiting examples of complement proteinsthat can be detected and/or measured singly or in any combinationaccording to the methods of this invention include C3, C9, factor B(FB), C5a, C5b, C3a, C8-alpha, C8-beta, C8-gamma, C7, C4, C6, membraneattack complex (MAC), mannose binding protein (MBP) and MBP-associatedserine protease 1 (MASP-1). These complement proteins can be detectedand/or measured singly or in any combination.

In some embodiments of this invention, the methods described herein arecarried out using an immunoassay system. In some embodiments, themethods described herein are carried out using a point-of-contact (POC)rapid assay system. In some embodiments, the methods of this inventionare carried out using an enzyme-linked immunosorbent assay (ELISA). Insome embodiments, the methods of this invention are carried out using alateral flow system.

In one embodiment, the present invention provides a dry-strip capable ofwicking a fluid applied thereto by capillarity within the strip, saidstrip comprising, in an upstream (at a first end) to downstream (at asecond end) direction and in the following order: 1) asample-application zone, 2) a reaction zone, and 3) a detection zone,wherein said reaction zone comprises a non-immobilized labeled firstantibody specific against an epitope of a complement C9 protein,effective to form therewith, a mobile complement C9 protein/antibodycomplex, and said detection zone comprises an immobilized secondantibody specific against an epitope of a complement C9 protein in saidcomplex, wherein said first and second antibodies are specific fordifferent epitopes on the same complement C9 protein and wherein, afterapplication of a body fluid sample to the sample-application zone at thefirst end, (i) sample migrates in a downstream direction on the striptoward the reaction zone, (ii) complement C9 protein in the samplereacts with the first antibody previously present in the reaction zoneto form a mobile, labeled complement C9 protein/antibody complex, (iii)the mobile, labeled complement C9 protein/antibody complex migratestoward the detection zone at the second end, (iv) the mobile, labeledcomplement C9 protein/antibody complex binds the immobilized secondantibody previously present in the detection zone, thereby immobilizingsaid complex in the detection zone. In some embodiments of the dry-stripof this invention, the first antibody and second antibody are present onthe dry-strip prior to application of the body fluid sample to thedry-strip. In some embodiments the first antibody is a monoclonalantibody and in some embodiments the second antibody is a monoclonalantibody. In some aspects, both the first antibody and the secondantibody are monoclonal antibodies.

In one embodiment, the present invention provides a dry-strip capable ofwicking a fluid applied thereto by capillarity within the strip, saidstrip comprising, in an upstream (at a first end) to downstream (at asecond end) direction and in the following order: 1) asample-application zone, 2) a reaction zone, and 3) a detection zone,wherein said reaction zone comprises a non-immobilized labeled firstantibody specific against an epitope of a complement C3 protein,effective to form therewith, a mobile complement C3 protein/antibodycomplex, and said detection zone comprises an immobilized secondantibody specific against an epitope of a complement C3 protein in saidcomplex, wherein said first and second antibodies are specific fordifferent epitopes on the same complement C3 protein and wherein, afterapplication of a body fluid sample to the sample-application zone at thefirst end, (i) sample migrates in a downstream direction on the striptoward the reaction zone, (ii) complement C3 protein in the samplereacts with the first antibody previously present in the reaction zoneto form a mobile, labeled complement C3 protein/antibody complex, (iii)the mobile, labeled complement C3 protein/antibody complex migratestoward the detection zone at the second end, (iv) the mobile, labeledcomplement C3 protein/antibody complex binds the immobilized secondantibody previously present in the detection zone, thereby immobilizingsaid complex in the detection zone. In some embodiments of the dry-stripof this invention, the first antibody and second antibody are present onthe dry-strip prior to application of the body fluid sample to thedry-strip. In some embodiments the first antibody is a monoclonalantibody and in some embodiments the second antibody is a monoclonalantibody. In some aspects, both the first antibody and the secondantibody are monoclonal antibodies.

In one embodiment, the present invention provides a dry-strip capable ofwicking a fluid applied thereto by capillarity within the strip, saidstrip comprising, in an upstream (at a first end) to downstream (at asecond end) direction and in the following order: 1) asample-application zone, 2) a reaction zone, and 3) a detection zone,wherein said reaction zone comprises a non-immobilized labeled firstantibody specific against an epitope of a complement factor B (FB)protein, effective to form therewith, a mobile complement FBprotein/antibody complex, and said detection zone comprises animmobilized second antibody specific against an epitope of a complementFB protein in said complex, wherein said first and second antibodies arespecific for different epitopes on the same complement FB protein andwherein, after application of a body fluid sample to thesample-application zone at the first end, (i) sample migrates in adownstream direction on the strip toward the reaction zone, (ii)complement FB protein in the sample reacts with the first antibodypreviously present in the reaction zone to form a mobile, labeledcomplement FB protein/antibody complex, (iii) the mobile, labeledcomplement FB protein/antibody complex migrates toward the detectionzone at the second end, (iv) the mobile, labeled complement FBprotein/antibody complex binds the immobilized second antibodypreviously present in the detection zone, thereby immobilizing saidcomplex in the detection zone. In some embodiments of the dry-strip ofthis invention, the first antibody and second antibody are present onthe dry-strip prior to application of the body fluid sample to thedry-strip. In some embodiments the first antibody is a monoclonalantibody and in some embodiments the second antibody is a monoclonalantibody. In some aspects, both the first antibody and the secondantibody are monoclonal antibodies.

In one embodiment, the present invention provides a dry-strip capable ofwicking a fluid applied thereto by capillarity within the strip, saidstrip comprising, in an upstream (at a first end) to downstream (at asecond end) direction and in the following order: 1) asample-application zone, 2) a reaction zone, and 3) a detection zone,wherein said reaction zone comprises a non-immobilized labeled firstantibody specific against an epitope of a complement MAC protein, whichcan be complement C5b, complement C6, complement C7, complement C8and/or complement C9, effective to form therewith, a mobile complementprotein/antibody complex, and said detection zone comprises animmobilized second antibody specific against an epitope of a complementMAC protein, which can be complement C5b, complement C6, complement C7,complement C8 and/or complement C9, in said complex, wherein said firstand second antibodies are specific for different epitopes on the samecomplement protein and wherein, after application of a body fluid sampleto the sample-application zone at the first end, (i) sample migrates ina downstream direction on the strip toward the reaction zone, (ii)complement protein in the sample reacts with the first antibodypreviously present in the reaction zone to form a mobile, labeledcomplement protein/antibody complex, (iii) the mobile, labeledcomplement protein/antibody complex migrates toward the detection zoneat the second end, (iv) the mobile, labeled complement protein/antibodycomplex binds the immobilized second antibody previously present in thedetection zone, thereby immobilizing said complex in the detection zone.In some embodiments of the dry-strip of this invention, the firstantibody and second antibody are present on the dry-strip prior toapplication of the body fluid sample to the dry-strip. In someembodiments the first antibody is a monoclonal antibody and in someembodiments the second antibody is a monoclonal antibody. In someaspects, both the first antibody and the second antibody are monoclonalantibodies.

In the embodiments of the dry-strip described herein, the dry-strip canbe configured to detect or measure a single complement protein in thesample (e.g., only C3, C5b, C6, C7, C8, C9, MAC or FB) or configured todetect or measure two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.)complement proteins (e.g., C3 and C9, C3 and FB, C9 and FB, C3 and C9and FB, C3 and one or more MAC proteins, FB and one or more MACproteins, two or more of any of the MAC proteins C5b, C6, C7, C8 and C9)in any combination. The two or more complement proteins can be detectedon the dry-strip in series or in parallel. For example, multipledry-strips can be arranged in parallel and/or multiple test lines can bearranged in series on a single dry-strip.

In further embodiments, the present invention provides a lateral flowimmunoassay device for detecting a marker, comprising: a membrane strip;a detecting antibody that binds a first epitope of the marker; a testline comprising a capturing antibody that binds a second epitope of themarker; and a control line comprising an antibody that binds a controlanalyte, wherein the marker is selected from the group consisting ofcomplement protein C3, complement protein factor B, complement MAC,complement C5b, complement C6, complement C7, complement C8 and/orcomplement protein C9, singly or in any combination.

In addition, the present invention provides a lateral flow immunoassaydevice comprising: a membrane strip; a first detecting antibody thatbinds a first epitope of complement C9; a first test line comprising afirst capturing antibody that binds a second epitope of complement C9; asecond detecting antibody that binds a first epitope of complement C3; asecond test line comprising a second capturing antibody that binds asecond epitope of complement C3; and at least one control linecomprising an antibody that binds a control analyte.

Further provided herein is a lateral flow immunoassay device comprising:a membrane strip; a first detecting antibody that binds a first epitopeof complement C9; a first test line comprising a first capturingantibody that binds a second epitope of complement C9; a seconddetecting antibody that binds a first epitope of complement factor B; asecond test line comprising a second capturing antibody that binds asecond epitope of complement factor B; and at least one control linecomprising an antibody that binds a control analyte.

The present invention also provides a lateral flow immunoassay devicecomprising: a membrane strip; a first detecting antibody that binds afirst epitope of complement C9; a first test line comprising a firstcapturing antibody that binds a second epitope of complement C9; asecond detecting antibody that binds a first epitope of complement C3; asecond test line comprising a second capturing antibody that binds asecond epitope of complement C3; a third detecting antibody that binds afirst epitope of complement factor B, a third test line comprising athird capturing antibody that binds a second epitope of complementfactor B; and at least one control line comprising an antibody thatbinds a control analyte.

In additional embodiments, the present invention provides a dry-stripcapable of wicking a fluid applied thereto by capillarity within thestrip, said strip comprising, in an upstream (at a first end) todownstream (at a second end) direction and in the following order: 1) asample-application zone, 2) a reaction zone, and 3) a detection zone,wherein said reaction zone comprises a non-immobilized labeled firstantibody specific against an epitope of a complement MAC protein (e.g.,C5b, C6, C7, C8 and/or C9, effective to form therewith, a mobilecomplement MAC/antibody complex, and said detection zone comprises animmobilized second antibody specific against an epitope of a complementMAC in said complex, wherein said first and second antibodies arespecific for different epitopes on the same complement MAC and wherein,after application of a body-fluid sample to the sample-application zoneat the first end, (i) sample migrates in a downstream direction on thestrip toward the reaction zone, (ii) complement MAC in the sample reactswith the first antibody previously present in the reaction zone to forma mobile, labeled complement MAC/antibody complex, (iii) the mobile,labeled complement MAC/antibody complex migrates toward the detectionzone at the second end, (iv) the mobile, labeled complement MAC/antibodycomplex binds the immobilized second antibody previously present in thedetection zone, thereby immobilizing said complex in the detection zone.

Also provided herein is a lateral flow immunoassay device for detectinga marker, comprising: a membrane strip; a detecting antibody that bindsa first epitope of the marker; a test line comprising a capturingantibody that binds a second epitope of the marker; and a control linecomprising an antibody that binds a control analyte, wherein the markeris selected from the group consisting of complement protein C3,complement protein factor B, complement MAC, complement protein C9 andany combination thereof. Such a lateral flow immunoassay device cancomprise a detecting antibody that comprises a label that provides asignal, e.g., for detection and/or quantitation, as is well known in theart.

In further embodiments, the present invention provides a lateral flowimmunoassay device comprising: a membrane strip; a first detectingantibody that binds a first epitope of a first protein selected from thegroup consisting of complement C9, complement C3, complement proteinfactor B and complement MAC; a first test line comprising a firstcapturing antibody that binds a second epitope of the first protein; asecond detecting antibody that binds a first epitope of a second proteinselected from the group consisting of complement C9, complement C3,complement protein factor B and complement MAC, wherein said secondprotein is different from said first protein; a second test linecomprising a second capturing antibody that binds a second epitope ofthe second protein; and at least one control line comprising an antibodythat binds a control analyte.

In some embodiments, the lateral flow immunoassay device describedherein can further comprise a third detecting antibody that binds afirst epitope of a third protein selected from the group consisting ofcomplement C9, complement C3, complement protein factor B and complementMAC, wherein said third protein is different from said first protein andsaid second protein, and a third test line comprising a third capturingantibody that binds a second epitope of the third protein.

In yet further embodiments, the lateral flow immunoassay devicedescribed herein can comprise a fourth detecting antibody that binds afirst epitope of a fourth protein selected from the group consisting ofcomplement C9, complement C3, complement protein factor B and complementMAC (e.g., C5b, C6, C7, C8 and/or C9), wherein said fourth protein isdifferent from said first protein and said second protein and said thirdprotein, and a fourth test line comprising a fourth capturing antibodythat binds a second epitope of the forth protein.

The lateral flow immunoassay device described herein can comprisedetecting antibodies wherein each respective detecting antibody comprisea label that provides a different signal that distinguishes each of saiddetecting antibodies from one another.

Furthermore, the present invention provides a method of monitoring asubject who is receiving treatment for bacterial meningitis, comprising:(a) obtaining serial samples of cerebrospinal fluid (CSF) from thesubject; (b) determining a level of complement C9, complement C3,complement MAC, which can be C5b, C6, C6, C8 and/or C9; and/orcomplement factor B in each of said samples; (c) comparing the level ofcomplement C9, complement C3, complement MAC, which can be C5b, C6, C6,C8 and/or C9; and/or complement factor B in the serial samples to detecta change in the level of complement C9, complement C3, complement MAC,which can be C5b, C6, C6, C8 and/or C9; and/or complement factor B overtime; and (d) modifying treatment of the subject, based on the resultsof the comparing step (c). Such modifying can include, for example,initiating treatment, enhancing treatment, resuming treatment,maintaining treatment, reducing treatment, halting treatment, etc.,pursuant to the results of the comparison. For example, in someembodiments, an increase over time would lead to initiating treatment,enhancing treatment, resuming treatment, or maintaining treatment. Insome embodiments, a decrease over time would lead to reducing treatmentor halting treatment.

For example, the clinician may detect a decrease in one or morecomplement proteins of this invention in a sample from the subjectduring treatment of the subject. Accordingly, the clinician may thenmodify the subject's treatment by adjusting the dosing of medicationsadministered, such as antibiotics, anti-inflammatory agents and/orcomplement inhibitors, or by discontinuing treatment once complementlevels have returned to normal.

In other embodiments, the clinician may detect an increase in one ormore (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) complement proteins ofthis invention in a sample from a subject during treatment of thesubject. Accordingly, the clinician may then modify the subject'streatment regimen, e.g., by increasing the dosage of medications, suchas antibiotics, anti-inflammatory agents and/or complement inhibitors,until a desired stabilization or decrease in complement protein level(s)is achieved. If no change in complement protein level is detected in asample from the subject during treatment, the clinician may modify thesubject's treatment regimen or may maintain the subject's treatmentregimen until a change in complement protein levels is observed.

A lateral flow immunoassay device embodiment of the present invention isdescribed herein and is comprised of a cellulose membrane strip, uponwhich is disposed a sample pad to absorb the sample fluid and allowgradual migration of the sample-and-particle-conjugate immune complexes,a wick at the distal end of the strip that absorbs the liquid sample andconjugate material to facilitate capillary migration through thecellulose membrane strip, and a particle conjugate pad comprising adetecting antibody bound to a label, or detection conjugate. Thecellulose membrane strip is the test zone region, upon which is disposeda test line, comprising monoclonal or polyclonal antibodies striped forcapturing the detection conjugate and a control line, comprising anantibody that binds a control analyte, such as IgG, and indicates to theuser that the test was successfully run. The lateral flow immunoassayfurther comprises a polyester film backing attached to the cellulosemembrane strip, and a pressure-sensitive laminate film backing. Eachlateral flow immunoassay may be packaged in a MYLAR zero-vapor barrierpouch, for example.

When a test sample is applied to the sample pad, the sample migratesfrom the sample pad through the particle conjugate pad, where any targetanalyte present will bind to the detecting antibody conjugate. Thesample then continues to migrate across the membrane until it reachesthe test line where the target/conjugate complex will bind to theimmobilized antibodies producing a visible line on the membrane. Thesample then migrates further along the membrane strip until it reachesthe control line, where excess antibody conjugate that did not bind thetest line will bind the control line and produce a second visible lineon the membrane. The control line ligand is often an antibody againstthe Fc region of the conjugated antibody. This control line indicatesthat the sample has migrated across the membrane as intended.

In certain embodiments, a lateral flow immunoassay device in accordancewith the present invention comprises a single membrane strip for thedetection of a single analyte. In other embodiments, the lateral flowimmunoassay detects two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.)analytes. When the lateral flow immunoassay detects two or moreanalytes, the test can be configured with multiple membrane stripsarranged in parallel or with multiple test lines arranged in series on asingle membrane strip.

In some embodiments, the lateral flow immunoassay is configured to testfor two analytes in a single test cassette in parallel. In someembodiments, the lateral flow immunoassay comprises two ports forinstilling the test samples and a separate membrane strip for eachanalyte. In other embodiments, the lateral flow immunoassay comprisesone port for instilling the sample and a separate membrane strip foreach analyte.

In some embodiments, the lateral flow immunoassay device may beconfigured to test for three analytes in a single test cassette inparallel. In some embodiments, the lateral flow immunoassay may comprisethree ports for instilling the test sample and a separate membrane stripfor each analyte. In other embodiments, the lateral flow immunoassay maycomprise one port for instilling the test sample and a separate membranestrip for each analyte.

In certain embodiments, the lateral flow immunoassay device may beconfigured to test for multiple analytes in a single test cassette inseries, e.g. a test cassette comprising a membrane strip with two testlines and one control line arranged in series or a test cassettecomprising a membrane strip with three test lines and one control linearranged in series.

The lateral flow immunoassay device presently disclosed may providequalitative and/or quantitative detection of the target markers.Qualitatively, two clear lines on the membrane may represent a positiveresult, whereas a single line in the control zone may represent anegative result. Quantitatively, the level or amount of the targetmarkers can be determined in samples based on comparison with standardcurves developed individually for each target and run prior to testsample assaying.

In some embodiments, the detecting antibody comprises a label thatprovides a signal that can be read visually by a clinician orelectronically via a commercial reader or electronically via an internalreader array on a printed circuit board installed within a lateral flowassay cassette. Various labels are suitable for use in the instantlydisclosed assays. In a specific embodiment, the label may be colloidalgold.

One skilled in the art will appreciate that various control analytes aresuitable for use in the methods of the instant invention to provideverification that the assay was successfully completed. In oneembodiment, the control analyte is IgG.

In some embodiments of the present methods, it is desirable to have alateral flow immunoassay that can detect more than complement protein ina single assay. For example, a dual lateral flow immunoassay that canqualitatively and quantitatively detect C9 in addition to C3, MAC or FBin the same aliquot of a body fluid may be highly desirable. Hence, inanother embodiment, a lateral flow immunoassay for the point-of-caredetection of complement proteins in a body fluid sample comprisingcomplement proteins is provided, the lateral flow immunoassay devicecomprising: a membrane strip; a first detecting antibody that binds afirst epitope of complement C9; a first test line comprising a firstcapturing antibody that binds a second epitope of complement C9; asecond detecting antibody that binds a first epitope of C3, MAC or FB; asecond test line comprising a second capturing antibody that binds asecond epitope of C3 or FB; and at least one control line comprising anantibody that binds a control analyte.

In some embodiments a lateral flow immunoassay that can qualitativelyand quantitatively detect C9 in addition to C3, MAC and FB in the samealiquot of a body fluid may be highly desirable. Hence, in anotherembodiment, a lateral flow immunoassay device for the point-of-caredetection of complement proteins in a body fluid sample comprisingcomplement proteins is provided, the lateral flow immunoassay devicecomprising: a membrane strip; a first detecting antibody that binds afirst epitope of complement C9; a first test line comprising a firstcapturing antibody that binds a second epitope of complement C9; asecond detecting antibody that binds a first epitope of C3; a secondtest line comprising a second capturing antibody that binds a secondepitope of C3; a third detecting antibody that binds a first epitope ofcomplement FB, a third test line comprising a third capturing antibodythat binds a second epitope of complement FB; and at least one controlline comprising an antibody that binds a control analyte. Additionaltest lines can be included comprising antibodies that bind complementMAC proteins, which can be complement C5b, complement C6, complement C7,complement C8 and/or complement C9. Such a lateral flow assay of thisinvention can comprise antibodies to complement C3, complement C5b,complement C6, complement C7, complement C8, complement C9 and/orcomplement FB in any combination and in any order of first antibodies,second antibodies, third antibodies, etc. in the appropriateconfiguration as would be known to one of ordinary skill in the art.

There is a wide variety of labels that may be used with a binding moiety(antibody or antigen) to form a labeled reagent. The choice of the labeldepends on the sensitivity required, ease of conjugation with thebinding moiety, stability requirements, available instrumentation, anddisposal provisions. Labels of the present invention may be soluble orparticulate, metallic, organic, or inorganic, and may include spectrallabels such as green fluorescent protein, fluorescent dyes (e.g.,fluorescein and its derivatives, rhodamine and its derivatives, biotin,avidin, and streptavidin), chemiluminescent compounds (e.g., luciferinand luminol); and enzymes (e.g., horseradish peroxidase, alkalinephosphatase, etc.), spectral colorimetric labels such as colloidal gold,or carbon particles, or colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads.

The label can be coupled directly or indirectly to a component of thebinding moiety according to methods well known in the art, such as thosedescribed in U.S. Pat. Nos. 4,863,875 and 4,373,932, each of which isincorporated herein by reference. Non-radioactive labels are oftenattached by indirect means. Generally, a ligand molecule (e.g., biotin)is covalently bound to the binding moiety. The ligand then binds to ananti-ligand (e.g., streptavidin) molecule which is either inherentlydetectable or is covalently bound to a signal system such as adetectable enzyme, a fluorescent compound, or a chemiluminescentcompound. The label may be attached to the binding moiety by a chemicallinker. Linker domains are typically polypeptide sequences, such aspoly-gly sequences of between about 5 and 200 amino acids. Preferredlinkers are often flexible amino acid sub-sequences. Such flexiblelinkers are known to persons skilled in the art. For example,poly(ethylene glycol) is available commercially (Shearwater Polymers,Inc. Huntsville, Ala.). The detection moiety can also be conjugateddirectly to the signal-generating compound, e.g., by conjunction with anenzyme or fluorophore.

The presence of a label can be detected by inspection, or a detectorthat monitors a particular probe or probe combination. Typical detectorsinclude spectrophotometers, phototubes and photodiodes, microscopes,scintillation counters, cameras, film and the like, as well ascombinations thereof. Examples of suitable detectors are widelyavailable from a variety of commercial sources known to persons skilledin the art.

For purposes of the present invention, preferred labels arenon-radioactive and are readily detected without the use ofsophisticated instrumentation. Preferably, the labels will yield avisible signal that is immediately discernable upon visual inspection,or by fluorescence detection. Preferred labels include those that may beobserved as: 1) chemiluminescence (using horseradish peroxidase and/oralkaline phosphatase with substrates that produce photons as breakdownproducts); 2) color change (colloidal gold, which produces a coloredprecipitate with the immuno-reactive event), and 3) fluorescence (using,e.g., fluorescein, and other fluorescent tags). In one preferredembodiment of the invention, colloidal gold is used as the label and thelabel is directly conjugated to the binding moiety (the antibody orantigen). When gold is used as the label, the reaction of labeledreagent-analyte complex with the capture reagent results in theappearance of a red colored deposit. As will be appreciated by one ofskill in the art, the color that appears upon the reaction of thecomplex with the capture reagent immobilized at the capturing zone willdepend on the label used.

In one embodiment of an assay format of the invention, the capture andcontrol capture reagents are immobilized on a solid substrate. There area variety of solid supports known to the art, which are suitable for usewith the present invention. For instance, the solid support may bebeads, membranes (e.g., nitrocellulose), microtiter wells (e.g., PVC orpolystyrene), strings, plastic, strips, or any surface onto whichantibodies may be deposited or immobilized. In addition, a wide varietyof organic and inorganic polymers, both natural and synthetic, may beemployed as the material for the solid surface. Illustrative polymersinclude polyethylene, polypropylene, poly(4-methylbutene), polystyrene,polymethacrylate, poly(ethylene terephthalate), rayon, nylon, poly(vinylbutyrate), polyvinylidene difluoride (PVDF), silicones,polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, and thelike. Other materials that may be employed include paper, glasses,ceramics, metals, metalloids, semiconductive materials, cements or thelike. In addition, substances that form gels, such as proteins (e.g.,gelatins), lipopolysaccharides, silicates, agarose and polyacrylamidescan be used. Polymers, which form several aqueous phases, such asdextrans and polyalkylene glycols or surfactants, such as phospholipidsor long chain (12-24 carbon atoms) alkyl ammonium salts and the like arealso suitable.

The manner of linking a wide variety of compounds to various surfaces iswell known and is amply illustrated in the literature. See, for example,IMMOBILIZED ENZYMES, Ichiro Chibata, Halsted Press, New York, 1978, andCuatrecasas, 1970, the disclosures of which are incorporated herein byreference. The capturing and control reagents may be covalently bound ornon-covalently attached through nonspecific bonding. If covalent bondingbetween a compound and the surface is desired, the surface will usuallybe polyfunctional or be capable of being polyfunctionalized. Functionalgroups which may be present on the surface and used for linking caninclude carboxylic acids, aldehydes, amino groups, cyano groups,ethylenic groups, hydroxyl groups, mercapto groups and the like. Inaddition to covalent bonding, various methods for noncovalently bindingan assay component can be used. Noncovalent binding is typicallynonspecific absorption of a compound to the surface. Typically, thesurface is blocked with a second compound to prevent nonspecific bindingof labeled assay components.

In one embodiment of the invention, the capture and control reagents arenonspecifically absorbed on a nitrocellulose membrane and blocked by ablocking buffer (e.g., 0.5% BSA; 4% sucrose in PBS).

The present invention also provides a kit comprising a dry-strip orlateral flow assay cassette or composition of this invention, incombination with reagents and instructions for use.

Definitions

As used herein, “a,” “an” and “the” can mean one or more than one,depending on the context in which it is used. For example, “a” cell canmean one cell or multiple cells.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

Furthermore, the term “about,” as used herein when referring to ameasurable value such as an amount of a compound or agent of thisinvention, dose, time, temperature, and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of thespecified amount.

A “subject” of this invention includes, but is not limited to, anyanimal that is susceptible to meningitis, including for example, humans,non-human primates, horses, cows, cats, dogs, pigs, rats, and mice.Administration of the various compositions of this invention can beaccomplished by any of several different routes. In specificembodiments, the compositions can be administered intramuscularly,subcutaneously, intraperitoneally, intradermally, intranasally,intracranially, sublingually, intravaginally, intrarectally, orally, ortopically. The compositions herein may be administered via a skinscarification method, or transdermally via a patch or liquid. Thecompositions can be delivered subdermally in the form of a biodegradablematerial that releases the compositions over a period of time.

“Bacterial meningitis” means meningitis (i.e., inflammation of themeninges) caused by any bacterial species including but not limited toStreptococcus sp., Escherichia sp., Staphylococcus sp., Listeria sp.,Neisseria sp., Salmonella sp. Haemophilus sp., and other Gram-negativeand Gram-positive bacteria.

When bacteria are not causing the inflammation or if the bacterialorganism cannot be grown and identified in the laboratory, it is called“aseptic meningitis.” Thus, aseptic meningitis is typically due toinfection by agents other than bacteria, which can be, for example,Coxsackie viruses, echoviruses, adenovirus, enteroviruses,herpesviruses, mumps virus, rabies virus other unspecified viruses,cryptococcal infection or infection due to other fungal organisms andinfection due to parasites. Aseptic meningitis, also known as sterilemeningitis, has also been associated, in some cases, with autoimmunedisease, cancer, Lyme disease, or an adverse reaction to medication.Meningitis can also be caused by mycobacterial species and is consideredto be aseptic meningitis.

Symptoms of bacterial meningitis and aseptic meningitis in adults aresimilar and can include, but are not limited to fever, headache, stiffneck, decreased consciousness, seizures, eye sensitivity and/or pain,rash, dizzy spells and vomiting. In babies, the signs of meningitis maybe a fever, irritability that is difficult to calm, decreased appetite,rash, vomiting, and a shrill cry. Babies also may have a stiff body andbulging soft spots on the head that aren't caused by crying. Babies withmeningitis may cry when handled. Young children with meningitis may actlike they have the flu (influenza), cough, or have trouble breathing.Older adults and people with other medical conditions may have only aslight headache and fever. They may not feel well and may have littleenergy.

Thus, a “subject in need thereof” as described herein is a subject thathas symptoms of meningitis and/or has been diagnosed with meningitisand/or is suspected of having meningitis and/or has been exposed to orhad contact with other subjects with symptoms of meningitis and/or havebeen diagnosed with and/or are suspected of having meningitis.

The term “analyte” means any entity, particularly a chemical,biochemical or biological entity to be assessed, e.g., whose amount(e.g., concentration or mass), activity, composition, or otherproperty(ies) is/are to be detected, measured, quantified, evaluated,analyzed, etc. An “analyte” can be a single molecular species or can becomposed of multiple distinct molecular species.

The term “antibody” encompasses intact and/or full lengthimmunoglobulins of types IgA, IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgE,IgD, IgM, IgY, antigen-binding fragments or single chains of completeimmunoglobulins (e.g., single chain antibodies, Fab fragments, F(ab′)2fragments, Fd fragments, scFv (single-chain variable), and dAbfragments), and other proteins that include at least one antigen-bindingimmunoglobulin variable region, e.g., a protein that comprises animmunoglobulin variable region, e.g., a heavy (H) chain variable region(VH) and a light (L) chain variable region (VL). The light chains of anantibody may be of type kappa or lambda. An antibody may be polyclonalor monoclonal. A polyclonal antibody contains immunoglobulin moleculesthat differ in sequence of their complementarity determining regions(CDRs) and, therefore, typically recognize different epitopes of anantigen. Often a polyclonal antibody is derived from multiple differentB cell lines each producing an antibody with a different specificity. Apolyclonal antibody may be composed largely of several subpopulations ofantibodies, each of which is derived from an individual B cell line. Amonoclonal antibody is composed of individual immunoglobulin moleculesthat comprise CDRs with the same sequence, and, therefore, recognize thesame epitope (i.e., the antibody is monospecific). Often a monoclonalantibody is derived from a single B cell line or hybridoma. An antibodymay be a “humanized” antibody in which for example, a variable domain ofrodent origin is fused to a constant domain of human origin or in whichsome or all of the complementarity-determining region amino acids oftenalong with one or more framework amino acids are “grafted” from arodent, e.g., murine, antibody to a human antibody, thus retaining thespecificity of the rodent antibody.

“Body fluid” means any fluid in the body that may be assayed for thepresence of complement proteins. Body fluids include, but are notlimited to, whole blood, serum, plasma, urine, tears, saliva, woundexudate, broncheoalveolar lavage fluid, and cerebrospinal fluid (CSF).

“Control” refers to a sample having a known amount or level of acomplement protein. In some embodiments, the control has a complementprotein level comparable to that of an individual who does not havebacterial meningitis, such that a test sample having a complementprotein level that is increased compared to the control is indicative ofbacterial meningitis.

“Epitope” refers to the minimum portion of a molecule that is recognizedby, and thus determines the immunospecificity of, an antibody that bindsto such epitope. The term is also used herein to refer to the minimumportion of a molecule that is recognized by a non-antibody specificbinding agent.

“Label” refers to a detectable moiety that facilitates the direct orindirect detection and/or quantitative or relative measurement of amolecule to which it is attached. A detectable label or detectablemoiety often produces a signal such as fluorescence, chemiluminescence,radioactivity, color, magnetic or paramagnetic properties, etc., thatrenders it detectable, e.g., by the use of instruments that detectfluorescence, chemiluminescence, radioactivity, color, magnetic field,magnetic resonance, etc., or in some cases by visual inspection. Thelabel may be, e.g., fluorescent substance; pigment; chemiluminescent orluminescent substance; colored substance; magnetic substance; or anon-magnetic metal particle such as gold colloid. In one embodiment, thedetecting antibodies suitable for use in the instant methods and assaysare conjugated to a colloidal gold label, which provides a color signal.

“Neoepitope” refers to an epitope that is generated or becomesdetectable as a result of proteolytic cleavage of a complement componentor cleavage product.

In certain embodiments of the assays and methods disclosed herein, thecomplement present in the body fluid sample tested is not substantiallyactivated by the assay or method itself. “Not substantially activated,”as used in this context, means that the methods and assays of thepresent invention are substantially free of in vitro activation causedby the test methods and/or materials. In this way, false positive testresults are avoided, since the lateral flow immunoassay is rapid andrequires less sample manipulation, thus avoiding many of the stimulithat contribute to in vitro complement activation.

“Point-of-care,” as used herein, refers to a device or method that canbe used or carried out at the bedside, in a doctor's office, in ahealthcare or treatment facility or any place where a sample can beobtained from a subject to be tested. Point-of-care tests generally donot require shipping a sample to a laboratory for processing or theexpertise of a skilled laboratory technician. The point-of-care methodsand tests described herein allow a clinician to receive criticalinformation at the patient's bedside, or at the site of evaluation ortreatment, which can more efficiently and effectively direct patientcare.

“Reader” refers to an instrument suitable for the detecting of thesignal produced by the label. Various instruments are known in the artfor the detection of label signals in diagnostic testing. In oneembodiment of the present invention, the label is colloidal gold and thereader is an instrument suitable for the qualitative and/or quantitativedetection of the color signal produced by the label. Suitable readersare available commercially from a variety of vendors, including BioAssayWorks (Ijamsville, Md.), the ESE-Quant from Qiagen (Hilden, Germany),Easterline LRE (Nordlingen, Germany), and Detekt Biomedical (Austin,Tex.). In some specific embodiments, the reader is a handheld readerthat quantifies the amount or concentration of the complement protein(s)being evaluated.

As used herein, “serial” collection of samples refers to taking aseparate sample (e.g., a CSF sample) from a subject over a course oftime, e.g., minutes, hours, days, weeks, months, etc.)

As used herein, “effective amount” refers to an amount of a compositionor formulation of this invention that is sufficient to produce a desiredeffect, which can be a therapeutic effect. The effective amount willvary with the age, general condition of the subject, the severity of thecondition being treated, the particular agent administered, the durationof the treatment, the nature of any concurrent treatment, thepharmaceutically acceptable carrier used, and like factors within theknowledge and expertise of those skilled in the art. As appropriate, an“effective amount” in any individual case can be determined by one ofordinary skill in the art by reference to the pertinent texts andliterature and/or by using routine experimentation. (See, for example,Remington, The Science And Practice of Pharmacy (20th ed. 2000)).

The compositions of the present invention can also include othermedicinal agents, pharmaceutical agents, carriers, diluents,immunostimulatory cytokines, etc. Actual methods of preparing suchdosage forms are known, or will be apparent, to those skilled in thisart. A dosage regimen can be one or more doses hourly, daily, weekly,monthly, yearly, etc. as deemed necessary to achieve the desiredprophylactic and./or therapeutic effect to be achieved by administrationof a composition of this invention to a subject. The efficacy of aparticular dosage can be determined according to methods well known inthe art.

Alternatively, pharmaceutical formulations of the present invention maybe suitable for administration to the mucous membranes of a subject(e.g., via intranasal administration, buccal administration and/orinhalation). The formulations may be conveniently prepared in unitdosage form and may be prepared by any of the methods well known in theart.

Efficacy of the treatment methods of this invention can be determinedaccording to well known protocols for determining the outcome of atreatment of a disease or infection of this invention. Determinants ofefficacy of treatment, include, but are not limited to, overallsurvival, disease-free survival, improvement in symptoms, time toprogression and/or quality of life, etc., as are well known in the art.

“Treat” or “treating” or “treatment” refers to any type of action thatimparts a modulating effect, which, for example, can be a beneficialeffect, to a subject afflicted with a disorder, disease or illness,including improvement in the condition of the subject (e.g., in one ormore symptoms), delay or reduction in the progression of the condition,delay of the onset of the disorder, disease or illness, and/or change inany of the clinical parameters of a disorder, disease or illness, etc.,as would be well known in the art.

It is understood that the foregoing detailed description is given merelyby way of illustration and that modifications and variations may be madetherein without departing from the spirit and scope of the invention.

EXAMPLES Example 1 C3 and Factor B (FB) as Bacterial MeningitisBiomarkers

Bacterial meningitis is characterized by high mortality and a high rateof persistent neurological impairment. Rapid etiologic diagnosis isessential for the adequate clinical management of patients withbacterial meningitis. Unfortunately, non-specific clinical symptoms andearly laboratory findings often do not unequivocally differentiatebetween bacterial and aseptic meningitis. Therefore, the identificationof a single discriminating parameter would be of high value in thedifferential diagnosis of acute meningitis.

This invention provides a method of diagnosing bacterial versus asepticmeningitis based on changes in the cerebrospinal fluid (CSF) levels ofspecific complement proteins, including, but not limited to C3, factor b(FB), MAC, C5b, C6, C7, C8, and C9 compared to normal CSF levels. Theinvention is based on the discovery that the CSF level of manycomplement proteins, which is normally low (nanogram to microgramrange), increases one to two orders of magnitude or more in bacterial,but not aseptic or virally-induced meningitis. This increase allowsready discrimination between bacterial and aseptic meningitis using avariety of either quantitative or semi-quantitative assay platforms suchas enzyme linked immunoassay (ELISA), multiplex or lateral flow assays,etc.

There is currently no inexpensive, point of care (POC) assay availableto rapidly and accurately discriminate between bacterial and asepticmeningitis. The rapid assay system embodied in this invention could beused in clinical settings where CSF was drawn based on suspicion ofbacterial meningitis. In such a platform, the assay would be used 1-2million times per year in the United States, with comparable sizedmarkets in Europe and Asia for diagnostic purposes.

ELISA for C3 and FB was performed on remnant CSF samples obtained frompatients with confirmed bacterial or viral meningitis. It was found thatboth proteins were significantly elevated in bacterial meningitiscompared to viral meningitis and controls (FIG. 2 and Table 2).

CSF levels in patients with bacterial meningitis were 21-fold higherthan those in aseptic meningitis and almost 19-fold higher thatcontrols. Factor B CSF levels in bacterial meningitis were even moreelevated: 63-fold compared to aseptic meningitis and 53-fold compared tocontrols. Linear regression analysis comparing paired C3 and FB CSFlevels in bacterial meningitis demonstrated a significant correlationbetween the two proteins (r²=0.33, p=0.008), strongly suggesting commoninflammatory mechanism(s) leading to their increase. In addition, bothproteins discriminated between bacterial and viral meningitis withextremely high sensitivity and specificity (Table 2).

Currently discrimination between bacterial and aseptic meningitis istime-consuming and expensive. Gram staining, biochemistry and bacterialculture, currently the only tests for diagnosing bacterial meningitis,take hours to days for completion and have significant false positiverates. The current repertoire of laboratory tests used to diagnosebacterial meningitis costs at least $1000. In one embodiment of thepresent invention, a POC lateral flow assay would involve approximately10-15 minutes and cost, e.g., around $100. This provides significanttime and cost saving. Such a POC assay could, for example, be used inemergency rooms and free-standing clinics or healthcare facilitiesthroughout the world.

Example 2 C9 as a Bacterial Meningitis Biomarker

A separate, but integral part of the pathogen clearance, the terminalcomplement pathway includes many different proteins. One of these, C9,is critical for the formation of the membrane attack complex (MAC), alarge pore-forming complex capable of lysing host pathogens includingmany bacteria and enveloped viruses. The MAC is composed of fivecomplement proteins, C5b, C6, C7, C8 and multiple C9 molecules (rangingfrom 12-18 per MAC). C9 molecules in the MAC form the actual porestructure that inserts into bacterial membranes resulting in theirosmotic lysis.

C9 is distinct from C3 and FB as a biomarker for bacterial meningitisbecause its function is unrelated to and independent of both proteins.

C9 is an acute phase protein; i.e., its production is elevated inresponse to inflammation due to trauma or infection. C9 has been shownto be transported across the blood-brain barrier at low levels in theabsence of infection. This process likely increases significantly duringbacterial meningitis as a result of cytokine-mediated inflammation thatweakens the blood-brain barrier. C9 production by several cells types inthe central nervous system is increased in response to inflammation andinfection.

Unlike C3 and factor B, many viral infections lead to a down-regulationof C9 production, which is a strategy used by some viruses to limitcomplement-mediated host defense functions. This enhances the relevanceof C9 as a biomarker for bacterial meningitis since C9 levels are lesslikely to increase in viral infections, which should enhance thespecificity and sensitivity of the assays of the present invention.

These unique features of C9 immunobiology, when paired with the abilityof C3 and factor B to discriminate bacterial from aseptic meningitisprovide for a diagnostic assay with additive and/or synergisticsensitivity.

Thus, in some embodiments of this invention, a CSF sample can beobtained from a subject (e.g., a subject in need thereof) and tested todetermine an amount of C9 according to the methods and assays describedherein.

Example 3 MAC as a Bacterial Meningitis Biomarker

A separate, but integral part of the pathogen clearance, the terminalcomplement pathway includes many different proteins. One of these, MACis a large pore-forming complex capable of lysing host pathogensincluding many bacteria and enveloped viruses. The MAC is composed offive complement proteins, C5b, C6, C7, C8 and multiple C9 molecules(ranging from 12-18 per MAC). C9 molecules in the MAC form the actualpore structure that inserts into bacterial membranes resulting in theirosmotic lysis.

MAC is distinct from C3 and FB as a biomarker for bacterial meningitisbecause its function is unrelated to and independent of both proteins.

Components of the MAC are acute phase proteins; i.e., their productionis elevated in response to inflammation due to trauma or infection.Production of MAC subcomponents by several cells types in the centralnervous system and other cell types is increased in response toinflammation and infection.

These unique features of MAC immunobiology alone allow it todiscriminate bacterial from aseptic meningitis provide for a diagnosticassay. In addition, when paired with the ability of other complementcomponents such as C3, to discriminate bacterial from aseptic meningitisprovide for a diagnostic assay with additive and/or synergisticsensitivity.

Thus, in some embodiments of this invention, a CSF sample can beobtained from a subject (e.g., a subject in need thereof) and tested todetermine an amount of MAC (e.g., C5, C6, C6, C8 and/or C9) according tothe methods and assays described herein.

Example 4

In an exemplary embodiment, the present invention provides a method ofdiagnosing bacterial infection in subjects with indwelling shunts orextra-ventricular devices (EVDs) based on changes in the cerebrospinalfluid (CSF) levels of specific complement proteins, including, but notlimited to, C3, C5b-9 (MAC) and factor B as compared to control CSFlevels. This embodiment is based on the observation that the CSF levelof many complement proteins which are normally low (nanogram to lowmicrogram range), can increase several orders of magnitude in CSF ofconfirmed bacterial infection. This dramatic increase allows for a rapiddetermination of shunt/EVD infection/meningitis using a variety ofeither quantitative or semi-quantitative assay platforms such as ELISA,multiplex or lateral flow assays. There is currently no inexpensive,point-of-care (POC) assay available to rapidly and accurately diagnose ashunt/EVD infection.

Upon application of the invention to a rapid assay system, the assaycould be used in any clinical setting where CSF was drawn based onsuspicion of shunt/EVD infection. In such a platform, the assay would beused ˜1 million times per year in the U.S., with comparable-sizedmarkets in Europe and Asia for diagnostic purposes.

We performed ELISAs for MAC on CSF samples obtained from pediatricneurosurgery subjects with shunts or EVDs on suspicion of bacterialinfection. We found that MAC levels were markedly upregulated insubjects with confirmed bacterial infection compared to those with noinfection (control subjects) (FIGS. 3 and 5, Table 3). The mean MAC CSFlevels on the first draw of CSF in patients with shunt/EVD infections(n=3) were 100-fold higher than those without infection (n=24). Thesedata indicated that the MAC and potentially other complement proteins oractivation fragments have significant potential as diagnostic markersfor shunt/EVD infection.

Currently, diagnosis of shunt/EVD infections is time-consuming andexpensive. Gram staining, biochemistry, and bacterial culture, currentlythe only tests for diagnosing bacterial meningitis, take hours to daysfor completion and have significant false-positive rates. The currentrepertoire of laboratory tests used to determine if the CSF is infectedcosts at least $1,000. In some embodiments of the present invention,employing a POC lateral flow assay would be rapid (e.g., test resultscould be available in approximately 10-15 minutes and low cost (e.g.,around $100). This provides a significant time and cost savings for thepatient. In addition, a POC biomarker assay would allow for betterpatient management.

The invention once applied to a POC rapid assay method would havesignificant market potential. It could be used in operating rooms andclinics throughout the world.

Example 5

To determine the value of repeated monitoring of CSF from patients withconfirmed shunt infection, we assayed for the CSF levels of MAC atvarious times points after the initial tap for up to fifteen days. TheMAC CSF level on the initial tap was greater than 400 times the normallevel observed in non-infected patients (FIG. 4). Intravenous antibiotictherapy was instituted and the patient was repeatedly tapped to followthe effectiveness of the antibiotic treatment. The CSF levels of MAClevels declined over time as the patient improved and the infectioncleared. These data indicate that following the CSF level of MAC, andpossibly other complement activation components, may be clinicallyuseful in monitoring effectiveness of antibiotic and other treatmentmodalities in shunt infections.

Example 6 MAC and C3 as Indicators of Shunt Failure

In an exemplary embodiment, the present invention provides a method ofdiscriminating shunt failure (symptomatic ventricular enlargement, SVE)from bacterial infection and/or asymptomatic ventricular enlargement(AVE) in subjects with indwelling shunts or extra-ventricular devices(EVDs) based on changes in the cerebrospinal fluid (CSF) levels ofspecific complement proteins, including, but not limited to, C3, C5b-9(MAC) and factor B as compared to control CSF levels. This embodiment isbased on the observation that the CSF level of many complement proteinswhich are normally low (nanogram range), can increase 8-10-fold inpatients with SVE compared to those with AVE, but be several orders ofmagnitude lower than in CSF of confirmed bacterial infection. Thisdistinct range of complement levels allows for a rapid determination ofshunt/EVD failures versus infection/meningitis using a variety of eitherquantitative or semi-quantitative assay platforms such as ELISA,multiplex or lateral flow assays. There is currently no inexpensive,point-of-care (POC) assay available to rapidly and accurately diagnose ashunt failure.

Upon application of the invention to a rapid assay system, the assaycould be used in any clinical setting where CSF was drawn based onsuspicion of shunt failure. In such a platform, the assay would be used˜1 million times per year in the U.S., with comparable-sized markets inEurope and Asia for diagnostic purposes.

We performed ELISAs for MAC on CSF samples obtained from pediatricneurosurgery subjects with shunts or EVDs on suspicion of shunt failureor bacterial infection. We found that MAC levels were markedly lower insubjects with confirmed shunt failure compared to those with bacterialinfection (FIG. 6). The mean MAC CSF levels in CSF in patients withshunt failure (n=35) was 8-10-fold higher than those with AVE but30-40-fold lower than patients with bacterial infection (n=6). Thesedata indicated that the MAC has significant potential as diagnosticmarkers for shunt failures. Similarly we performed ELISAs for C3 on CSFsamples obtained from pediatric neurosurgery subjects with shunts orEVDs on suspicion of shunt failure or bacterial infection. We found thatC3 levels were significantly lower in subjects with confirmed shuntfailure compared to those with bacterial infection (FIG. 7). The mean C3CSF levels in CSF in patients with shunt failure (n=13) was 2-foldhigher than those with AVE but 3-fold lower than patients with bacterialinfection (n=3). These data indicated that the C3 has significantpotential as diagnostic markers for shunt failures.

Currently, diagnosis of shunt failure is time-consuming and expensive.Gram staining, biochemistry, and bacterial culture, to determine if thepatient has a low grade infection as opposed to shunt failure, takeshours to days for completion and have significant false-positive rates.The current repertoire of laboratory tests used to determine if the CSFis infected costs at least $1,000. Patients with suspected shunt failurealso undergo expensive imaging studies to determine if there isventricular enlargement characteristic or shunt failure. In someembodiments of the present invention, employing a POC lateral flow assaywould be rapid (e.g., test results could be available in approximately10-15 minutes and low cost (e.g., around $100). This provides asignificant time and cost savings for the patient. In addition, a POCbiomarker assay would allow for better patient management.

The invention once applied to a POC rapid assay method would havesignificant market potential. It could be used in operating rooms andclinics throughout the world.

All references cited herein, including non-patent publications, patentapplications, GenBank® Database accession numbers and patents, areincorporated by reference herein in their entireties to the same extentas if each was individually and specifically indicated to beincorporated by reference, and was reproduced in its entirety herein.

While particular embodiments of the present invention have beenillustrated and described, it would be understood by one skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is, therefore,intended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

TABLE 1 Antibodies to C3 Species Antigen Supplier mouse C3a Hycult(HM2075) mouse C3a Quidel (A203) chicken C3a GenTex (GTX78198) mouse C3aQuidel (A203) goat C3a SantaCruz (sc17237) mouse C3a Hycult (HM2073)mouse C3a Hycult (HM2074) chicken C3a Abcam (ab48580) mouse C3a Hycult(HM2073) mouse C3a Quidel (A203) chicken C3a Abcam (ab48580) mouse C3aHycult (HM2073) goat C3a SantaCruz (sc17237) rabbit C3d Abcam (ab15981)rabbit C3d Abcam (ab15981) rabbit C3d Abcam (ab15981) rabbit C3d Abcam(ab15981) rabbit C3d Abcam (ab15981) rabbit C3d Abcam (ab15981) goat C3MP Biomedicals (55237) goat C3 MP Biomedicals (55237) rabbit C3d Abcam(ab15981) rabbit C3d Abcam (ab15981) rabbit C3d Abcam (ab15981) rabbitC3d Abcam (ab15981) rabbit C3d Abcam (ab15981) rabbit C3d Abcam(ab15981) chicken C3a Abcam (ab48580) chicken C3a Abcam (ab48580) goatC3 MP Biomedicals (55237) goat C3 MP Biomedicals (55237) goat C3 MPBiomedicals (55237) mouse C3a Quidel (A2103) chicken C3a GenTex(GTX78198) goat C3a SantaCruz (sc17237) chicken C3a Abcam (ab48580)mouse C3a Hycult (HM2073) mouse C3a Hycult (HM2074) mouse iC3b Quidel(A209) mouse iC3b AbD serotec (MCA2607) mouse iC3b AbD serotec (MCA2607)mouse iC3b Quidel (A209) mouse iC3b Quidel (A209) mouse iC3b Quidel(A209) mouse iC3b Quidel (A209) rat iC3b Hycult (HM2199) rat iC3b Hycult(HM2199) rat iC3b Hycult (HM2199) rat iC3b Hycult (HM2199) rat iC3bHycult (HM2199) mouse active C3 Hycult (HM2168) mouse active C3 Hycult(HM2168) mouse active C3 Hycult (HM2257) mouse active C3 Hycult (HM2257)mouse C3 alpha Meridian (H54189M) mouse neo C3d Quidel (A250) goat C3 MPBiomedicals (55237) goat C3 MP Biomedicals (55237) rabbit C3d Abcam(ab15981) rabbit C3d Abcam (ab15981) rat C3d Hycult (HM2198) rat C3gHycult (HM2199) mouse neo C3d Quidel (A250) goat C3 MP Biomedicals(55237) mouse active C3 Hycult (HM2168) mouse Active C3 Hycult (HM2257)mouse iC3b Quidel (A209) mouse Neo C3d Quidel (A250) goat C3 MPBiomedicals (55237) rat C3g Hycult (HM 2199) goat C3 MP Biomedicals(55237) rat C3g Hycult (HM 2199) goat C3 MP Biomedicals (55237) rat C3gHycult (HM 2199)

TABLE 2 Statistical parameters for C3 and factor B in bacterial versusaseptic meningitis C3 Factor B BM AM Controls BM AM Controls Mean ± SE47 ± 10.8 2.2 ± 0.4 2.5 ± 0.3 15.7 ± 3.8 0.25 ± 0.04 0.3 ± 0.03 (μg/ml)Sensitivity 100% 100% Sencificity  95% 100% BM, bacterial meningitis,AM, aseptic meningitis

TABLE 3 Statistical parameters for MAC in shunt/EVD infections Shuntinfection Controls n 3 24 Mean ± SEM (ng/ml) 1581 ± 1218 15.6 ± 5 Range270-4014 0-97

1-9. (canceled)
 10. A method of guiding a treatment for an infectionassociated with an indwelling shunt and/or extra-ventricular device in asubject in need thereof, comprising: a) measuring the amount ofcomplement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8, and/or complement C9 in acerebrospinal fluid (CSF) sample obtained from the subject prior toadministration of the treatment for the infection associated with anindwelling shunt and/or extra-ventricular device; b) administering thetreatment to the subject; c) measuring the amount of complement C3,complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8, and/or complement C9 in a cerebrospinalfluid (CSF) sample obtained from the subject at one or more time pointsafter (b); d) guiding the treatment of the subject for the infectionassociated with an indwelling shunt and/or extra-ventricular deviceusing the measurement(s) of (c) such that an increase or no change inthe amount(s) measured in (c) relative to the amount(s) measured in (a)leads to a subsequent enhancement of the treatment, and a decrease inthe amount(s) measured in (c) relative to the amount(s) measured in (a)leads to no change or a subsequent reduction of the treatment. 11.(canceled)
 12. A method of treating a subject with an infectionassociated with an indwelling shunt and/or extra-ventricular device,comprising: a) measuring the amount of complement C3, complement factorB, complement MAC, complement C5b, complement C6, complement C7,complement C8, and/or complement C9 in a cerebrospinal fluid (CSF)sample obtained from the subject; b) comparing the amount of complementC3, complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8, and/or complement C9 measured in (a) withthe amount of complement C3, complement factor B, complement MAC,complement C5b, complement C6, complement C7, complement C8, and/orcomplement C9 measured in a control sample, wherein an amount ofcomplement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8, and/or complement C9measured in (a) that is greater than the amount of complement C3,complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8, and/or complement C9 measured in thecontrol sample identifies an infection in the subject associated withthe indwelling shunt and/or extra-ventricular device; and c) treatingthe subject identified in (b) for the infection associated with theindwelling shunt and/or extra-ventricular device.
 13. A method oftreating a subject with an infection associated with an indwelling shuntand/or extra-ventricular device, comprising: a) measuring the amount ofcomplement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8, and/or complement C9 in acerebrospinal fluid (CSF) sample obtained from the subject, wherein anamount of complement C3, complement factor B, complement MAC, complementC5b, complement C6, complement C7, complement C8, and/or complement C9measured in (a) that is greater than a threshold amount of complementC3, complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8, and/or complement C9 identifies aninfection in the subject associated with the indwelling shunt and/orextra-ventricular device; and b) treating the subject identified in (a)for the infection associated with the indwelling shunt and/orextra-ventricular device. 14-34. (canceled)
 35. The method of claim 10,wherein the amount of complement C9 and the amount of complement C3 inthe CSF sample are measured.
 36. The method of claim 10, wherein theamount of complement C9 and the amount of complement factor B in the CSFare measured.
 37. The method of claim 10, wherein the amount ofcomplement C9 and the amount of complement C5b in the CSF sample aremeasured.
 38. The method of claim 10, wherein the amount of complementC9 and the amount of complement C6 in the CSF sample are measured. 39.The method of claim 10, wherein the amount of complement C9 and theamount of complement C7 in the CSF sample are measured.
 40. The methodof claim 10, wherein the amount of complement C9 and the amount ofcomplement C8 in the CSF sample are measured.
 41. The method of claim10, wherein the amount of complement C8 and the amount of complement C3in the CSF sample are measured.
 42. The method of claim 10, wherein theamount of complement C8 and the amount of complement factor B in the CSFare measured.
 43. The method of claim 10, wherein the amount ofcomplement C8 and the amount of complement C5b in the CSF sample aremeasured.
 44. The method of claim 10, wherein the amount of complementC8 and the amount of complement C6 in the CSF sample are measured. 45.The method of claim 10, wherein the amount of complement C8 and theamount of complement C7 in the CSF sample are measured.
 46. The methodof claim 10, wherein the amount of complement C7 and the amount ofcomplement C3 in the CSF sample are measured.
 47. The method of claim10, wherein the amount of complement C7 and the amount of complementfactor B in the CSF are measured.
 48. The method of claim 10, whereinthe amount of complement C7 and the amount of complement C5b in the CSFsample are measured.
 49. The method of claim 10, wherein the amount ofcomplement C7 and the amount of complement C6 in the CSF sample aremeasured.
 50. The method of claim 10, wherein the amount of complementC6 and the amount of complement C3 in the CSF sample are measured. 51.The method of claim 10, wherein the amount of complement C6 and theamount of complement factor B in the CSF are measured.
 52. The method ofclaim 10, wherein the amount of complement C6 and the amount ofcomplement C5b in the CSF sample are measured.
 53. The method of claim10, wherein the amount of complement C5b and the amount of complement C3in the CSF sample are measured.
 54. The method of claim 10, wherein theamount of complement C5b and the amount of complement factor B in theCSF are measured.
 55. The method of claim 10, wherein the amount ofcomplement MAC and the amount of complement factor B and the amount ofcomplement C3 in the CSF are measured.
 56. The method of claim 10,wherein the amount of complement C9 and the amount of complement factorB and the amount of complement C3 in the CSF are measured.
 57. Themethod of claim 10, wherein the measuring is carried out with a point ofcontact (POC) rapid assay system.
 58. The method of claim 10, whereinthe measuring is carried out with a lateral flow system.
 59. The methodof claim 10, wherein the measuring is carried out with an immunoassay.60. The method of claim 10, wherein the measuring is carried out with anenzyme-linked immunosorbent assay (ELISA). 61-67. (canceled)
 68. Amethod of treating a subject with an infection associated with anindwelling shunt and/or extra-ventricular device, comprising treatingthe subject identified as having an amount of complement C3, complementfactor B, complement MAC, complement C5b, complement C6, complement C7,complement C8, and/or complement C9 that is greater than an amount ofcomplement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8, and/or complement C9 in acontrol sample.
 69. A method of treating a subject with an infectionassociated with an indwelling shunt and/or extra-ventricular device,comprising treating the subject identified as having an amount ofcomplement C3, complement factor B, complement MAC, complement C5b,complement C6, complement C7, complement C8, and/or complement C9measured in (a) that is greater than a threshold amount of complementC3, complement factor B, complement MAC, complement C5b, complement C6,complement C7, complement C8, and/or complement C9.